Compositions and methods for treating or ameliorating neuroinflammation, neurodegeneration, neuropathic pain, and migraine

ABSTRACT

In alternative embodiments, provided are methods for increasing levels of and/or upregulating the expression of ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) to treat, ameliorate, prevent, reverse, decrease the severity or duration of: a neuropathic pain, including an inflammation-induced neuropathic pain, a nerve or CNS inflammation, a, a post nerve injury pain, a post-surgical pain, a chemotherapeutic-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced allodynia) a neurodegeneration or neurodegenerative disease or condition, a migraine, and/or a hyperalgesia. In alternative embodiments, provided are methods comprising administering formulations and pharmaceutical compositions comprising an APOA1BP polypeptide or protein that is a human or a mammalian APOA1BP, or an AIBP1 or an AIBP2, or a recombinant, peptidomimetic or a synthetic APOA1BP, or a bioisostere of an ApoA-I Binding Protein to treat, ameliorate prevent, reverse, decrease the severity of a neuropathic pain, a TLR4-mediated allodynia and/or a hyperalgesia.

RELATED APPLICATIONS

This application is divisional patent application under 35 U.S.C. § 121to U.S. patent application Ser. No. 16/060,576, Jun. 8, 2018 (nowpending), which is a national phase application claiming benefit ofpriority under 35 U.S.C. § 371 to Patent Convention Treaty (PCT)International Application serial number PCT/US2016/064938, filed Dec. 5,2016, which claims priority under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Ser. No. 62/265,752, filed Dec. 10, 2015. The aforementionedapplications are expressly incorporated herein by reference in theirentirety and for all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under AR064194 andHL124174 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to medicine, pain control and cellbiology. In particular, in alternative embodiments, provided are methodsfor increasing levels of and/or upregulating the expression of ApoA-IBinding Protein (APOA1BP, AIBP, or AI-BP) to treat, ameliorate, prevent,reverse, decrease the severity and/or duration of: a neuropathic pain, aCNS inflammation, an allodynia, a post nerve injury pain, apost-surgical pain, a chemotherapeutic-induced peripheral neuropathy(CIPN) (e.g., cisplatin-induced allodynia) a neurodegeneration,including e.g., a neurodegenerative disease or condition such asAlzheimer's disease, a hyperalgesia, and/or primary headaches such asmigraines and cluster headaches. In alternative embodiments, providedare methods comprising administering formulations and pharmaceuticalcompositions comprising an APOA1BP polypeptide or protein that is ahuman or a mammalian APOA1BP, or a recombinant, peptidomimetic or asynthetic APOA1BP, or a bioisostere of an ApoA-I Binding Protein totreat, ameliorate prevent, reverse, decrease the severity of aneuropathic pain, an allodynia, a hyperalgesia, a neurodegenerativedisease or condition such as Alzheimer's disease, and/or a primaryheadache such as a migraine.

BACKGROUND

Apolipoprotein A-1 Binding Protein, or ApoA-I binding protein (AIBP),also called also called NAXE, NAD(P)HX epimerase, is a secreted proteindiscovered in a screen of proteins that physically associate with apoA-I(8). The human AIBP gene (APOA1BP) is located at 1q22. AIBP is asecreted protein. It has a presumed N-terminal signal peptide, whichlikely is cleaved off the protein during protein secretion from thecell.

Persistent pain states (greater than 3 months), arising frominflammatory disease (e.g., arthritis, 41 million people in the US;cancer, 8.5 million; and back pain, 6 million¹) have extraordinarynegative impact on quality of life. While opiates, NSAIDs, andanticonvulsants can relieve pain for short intervals, they are lesseffective for chronic therapy, particularly when components of the painstate involve persistent inflammation and/or injury to the peripheralnerve²⁻⁴. Aside from efficacy, many of the potent agents are beset withlimiting side effects and issues related to dependence and addiction⁵.This relative lack of long-term efficacy of even approved agents isevident from clinical trial results, which often indicate that mostsubjects complete even successful trials with pain that is sufficientlysevere as to permit reentry into the same trial⁶.

The pain states arising from local tissue injury or inflammationtypically show a time course that parallels the onset and resolution ofthe injury state⁷, whereas nerve injury leads to a persistent painstate. However, as reported in humans, there is a growing appreciationthat the pain originating from prolonged inflammation may persist evenwhen the inflammatory state resolves, i.e. neutrophils, macrophages orcytokines are no longer detected. Thus, following surgeries such asherniorrhaphies, arthroscopies and thoracotomies, up to 30% of thepopulations may show pain that last greater than 3 months^(8,9). In theclassic clinical example of persistent inflammation, rheumatoidarthritis is characterized by joint inflammation, joint remodeling, andpain. While the association of pain with inflammation is not unexpected,patients continue to report moderate to severe pain despite remission orshowing minimal inflammatory signs^(10,11), suggesting the developmentof a chronic pain state.

SUMMARY

In alternative embodiments, provided are methods and uses for treating,ameliorating, preventing, reversing or decreasing the severity orduration of:

-   -   a neuropathic pain,    -   an inflammation-induced neuropathic pain,        -   wherein optionally the inflammation-induced neuropathic pain            comprises a Toll-like receptor 4 (TLR4)-mediated            inflammation-induced neuropathic pain,    -   nerve or central nervous system (CNS) inflammation,        -   wherein optionally the nerve or CNS inflammation comprises a            TLR4-mediated nerve or CNS inflammation,    -   an allodynia,        -   wherein optionally the allodynia comprises a TLR4-mediated            allodynia, —a post nerve or tissue injury pain or            neuropathic pain,        -   wherein optionally the post nerve or tissue injury pain or            neuropathic pain is generated or caused by, or is a sequelae            to, trauma, chemotherapy, arthritis, diabetes, or viral            infection,    -   a post-surgical pain or neuropathic pain,    -   a chemotherapeutic-induced peripheral neuropathy (CIPN) (e.g., a        cisplatin-induced CIPN or allodynia),        -   wherein optionally the allodynia comprises a TLR4-mediated            allodynia,    -   a neurodegenerative disease or condition, optionally a chronic        or progressive neurodegenerative disease or condition,        optionally Alzheimer's disease or a Chronic Traumatic        Encephalopathy (CTE) or a related tauopathy, a traumatic brain        injury (TBI), a posttraumatic stress disorder, a traumatic war        neurosis, or a post-traumatic stress syndrome (PTSS),    -   a primary headache, optionally a migraine or a cluster headache,        and/or    -   a hyperalgesia,

in a subject by adding, increasing levels of and/or upregulating theexpression of an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP),wherein the method comprises:

(a) providing a formulation or a pharmaceutical composition comprising:

(i) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptidecompound or composition, or a compound that increases expression oractivity of, or encodes, a APOA1BP polypeptide or nucleic acid, or apolypeptide or peptide having an APOA1BP activity, or anAPOA1BP-stimulating compound or composition;

(ii) the formulation or pharmaceutical composition of (i), wherein thecompound that increases expression or activity of, or encodes, a APOA1BPpolypeptide is a nucleic acid that expresses or encodes a APOA1BPpolypeptide or a polypeptide having a APOA1BP polypeptide activity,

and optionally a APOA1BP-stimulating compound or composition increasesor stimulates (activates) the activity of a APOA1BP promoter ortranscriptional regulatory sequence or motif,

and optionally the nucleic acid that expresses or encodes a APOA1BPpolypeptide or a polypeptide having a APOA1BP polypeptide activity iscontained in an expression vehicle, vector, recombinant virus, orequivalent, and optionally the vector or virus is or comprises anadenovirus vector or an adeno-associated virus (AAV) vector, aretrovirus, a lentiviral vector, a herpes simplex virus, a humanimmunodeficiency virus (HIV), or a synthetic vector,

and optionally the AAV vector comprises or is:

an adeno-associated virus (AAV), or an adenovirus vector,

an AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ/8™(Cell Biolabs, Inc., San Diego, Calif.),

a rhesus-derived AAV, or the rhesus-derived AAV AAVrh.10hCLN2,

an AAV capsid mutant or AAV hybrid serotype,

an organ-tropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41mutant,

wherein optionally the AAV is engineered to increase efficiency intargeting a specific cell type that is non-permissive to a wild type(wt) AAV and/or to improve efficacy in infecting only a cell type ofinterest,

and optionally the hybrid AAV is retargeted or engineered as a hybridserotype by one or more modifications comprising: 1) a transcapsidation,2) adsorption of a bi-specific antibody to a capsid surface, 3)engineering a mosaic capsid, and/or 4) engineering a chimeric capsid;

(iii) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducingcompound or composition;

(iv) the formulation or pharmaceutical composition of any of (i) to(iii), wherein the an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)polypeptide or protein is a human or a mammalian APOA1BP, or a AIBP1 ora AIBP2, or a recombinant, peptidomimetic or a synthetic APOA1BP, or abioisostere of an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP);

(v) the formulation or pharmaceutical composition of any of (i) to (iv),formulated for administration in vivo; or formulated for enteral orparenteral administration, or for oral, subcutaneous (SC), intramuscular(IM), intravenous (IV) or intrathecal (IT) administration,

wherein optionally the formulation or pharmaceutical composition, or therecombinant, peptidomimetic or a synthetic APOA1BP, or bioisostere ofAPOA1BP, or nucleic acid encoding the APOA1BP, or vector havingcontained therein a nucleic acid encoding the APOA1BP, is carried in ananoparticle, a particle, a micelle or a liposome or lipoplex, apolymersome, a polyplex or a dendrimer, which optionally can furthercomprise or express a cell or CNS penetrating moiety or peptide or a CNStargeting moiety or peptide; or

(vi) the formulation or pharmaceutical composition of any of (i) to (v),formulated for as a nanoparticle, a liposome, a tablet, a pill, acapsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, anaerosol, a spray, a lozenge, an aqueous or a sterile or an injectablesolution, or an implant (e.g., an intrathecal implant); and

(b) administering the formulation or the pharmaceutical composition of(a) to a subject in need thereof, wherein optionally the subject is ahuman or an animal,

thereby treating, ameliorating, preventing, reversing or decreasing theseverity or duration of the:

-   -   neuropathic pain,    -   inflammation-induced neuropathic pain,        -   wherein optionally the inflammation-induced neuropathic pain            comprises a Toll-like receptor 4 (TLR4)-mediated            inflammation-induced neuropathic pain,    -   inflammation-induced neuropathic pain,    -   nerve or CNS inflammation,        -   wherein optionally the nerve or CNS inflammation comprises a            TLR4-mediated nerve or CNS inflammation,    -   allodynia,        -   wherein optionally the allodynia comprises a TLR4-mediated            allodynia,    -   a post nerve or tissue injury pain or neuropathic pain,        -   wherein optionally the post nerve or tissue injury pain or            neuropathic pain is generated or caused by, or is a sequelae            to, trauma, chemotherapy, arthritis, diabetes, or viral            infection,    -   post-surgical pain or neuropathic pain,    -   chemotherapeutic-induced peripheral neuropathy (CIPN) (e.g., a        cisplatin-induced CIPN or allodynia),    -   a neurodegenerative disease or condition, optionally a chronic        or progressive neurodegenerative disease or condition,        optionally Alzheimer's disease or a Chronic Traumatic        Encephalopathy (CTE) or a related tauopathy, a traumatic brain        injury (TBI), a posttraumatic stress disorder, a traumatic war        neurosis, or a post-traumatic stress syndrome (PTSS),    -   a primary headache, optionally a migraine or a cluster headache,        and/or    -   hyperalgesia.

In alternative embodiments, provided are kits comprising a formulationor a pharmaceutical composition used in a method or use as providedherein, and optionally comprising instructions on practicing a method oruse as provided herein.

In alternative embodiments, provided are Uses of a formulation or apharmaceutical composition as used in a method provided herein, in themanufacture of a medicament.

In alternative embodiments, provided are Uses of a formulation or apharmaceutical composition as used in a method provided herein, in themanufacture of a medicament for treating, ameliorating, preventing,reversing and/or decreasing the severity or duration of: —neuropathicpain,

-   -   inflammation-induced neuropathic pain, wherein optionally the        inflammation-induced neuropathic pain comprises a Toll-like        receptor 4 (TLR4)-mediated inflammation-induced neuropathic        pain,    -   inflammation-induced neuropathic pain,    -   nerve or CNS inflammation,        -   wherein optionally the nerve or CNS inflammation comprises a            TLR4-mediated nerve or CNS inflammation,    -   allodynia,        -   wherein optionally the allodynia comprises a TLR4-mediated            allodynia,    -   a post nerve or tissue injury pain or neuropathic pain,        -   wherein optionally the post nerve or tissue injury pain or            neuropathic pain is generated or caused by, or is a sequelae            to, trauma, chemotherapy, arthritis, diabetes, or viral            infection,    -   post-surgical pain or neuropathic pain,    -   chemotherapeutic-induced peripheral neuropathy (CIPN) (e.g., a        cisplatin-induced CIPN or allodynia),    -   a neurodegenerative disease or condition, optionally a chronic        or progressive neurodegenerative disease or condition,        optionally Alzheimer's disease or a Chronic Traumatic        Encephalopathy (CTE) or a related tauopathy, a traumatic brain        injury (TBI), a posttraumatic stress disorder, a traumatic war        neurosis, or a post-traumatic stress syndrome (PTSS),    -   a primary headache, optionally a migraine or a cluster headache,        and/or    -   hyperalgesia

In alternative embodiments, provided are formulations, pharmaceuticalcompositions or therapeutic combinations for use in a method fortreating, ameliorating, preventing, reversing or decreasing the severityand/or duration of:

-   -   neuropathic pain,    -   inflammation-induced neuropathic pain,        -   wherein optionally the inflammation-induced neuropathic pain            comprises a Toll-like receptor 4 (TLR4)-mediated            inflammation-induced neuropathic pain,    -   inflammation-induced neuropathic pain,    -   nerve or CNS inflammation,        -   wherein optionally the nerve or CNS inflammation comprises a            TLR4-mediated nerve or CNS inflammation,    -   allodynia,        -   wherein optionally the allodynia comprises a TLR4-mediated            allodynia,    -   a post nerve or tissue injury pain or neuropathic pain,        -   wherein optionally the post nerve or tissue injury pain or            neuropathic pain is generated or caused by, or is a sequelae            to, trauma, chemotherapy, arthritis, diabetes, or viral            infection,    -   post-surgical pain or neuropathic pain,    -   chemotherapeutic-induced peripheral neuropathy (CIPN) (e.g., a        cisplatin-induced CIPN or allodynia),    -   a neurodegenerative disease or condition, optionally a chronic        or progressive neurodegenerative disease or condition,        optionally Alzheimer's disease or a Chronic Traumatic        Encephalopathy (CTE) or a related tauopathy, a traumatic brain        injury (TBI), a posttraumatic stress disorder, a traumatic war        neurosis, or a post-traumatic stress syndrome (PTSS),    -   a primary headache, optionally a migraine or a cluster headache,        and/or    -   hyperalgesia.

wherein the formulation or a therapeutic combination comprises one of,all or several of:

-   -   (i) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)        polypeptide compound or composition, or a compound that        increases expression or activity of, or encodes, a APOA1BP        polypeptide or nucleic acid, or a polypeptide or peptide having        an APOA1BP activity, or an APOA1BP-stimulating compound or        composition;    -   (ii) the formulation or pharmaceutical composition of (i),        wherein the compound that increases expression or activity of,        or encodes, a APOA1BP polypeptide is a nucleic acid that        expresses or encodes a APOA1BP polypeptide or a polypeptide        having a APOA1BP polypeptide activity,    -   and optionally a APOA1BP-stimulating compound or composition        increases or stimulates (activates) the activity of a APOA1BP        promoter or transcriptional regulatory sequence or motif,

and optionally the nucleic acid that expresses or encodes a APOA1BPpolypeptide or a polypeptide having a APOA1BP polypeptide activity iscontained in an expression vehicle, vector, recombinant virus, orequivalent, and optionally the vector or virus is or comprises anadenovirus vector or an adeno-associated virus (AAV) vector, aretrovirus, a lentiviral vector, a herpes simplex virus, a humanimmunodeficiency virus (HIV), or a synthetic vector,

and optionally the AAV vector comprises or is:

an adeno-associated virus (AAV), or an adenovirus vector,

an AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ/8™(Cell Biolabs, Inc., San Diego, Calif.),

a rhesus-derived AAV, or the rhesus-derived AAV AAVrh.10hCLN2,

an AAV capsid mutant or AAV hybrid serotype,

an organ-tropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41mutant,

wherein optionally the AAV is engineered to increase efficiency intargeting a specific cell type that is non-permissive to a wild type(wt) AAV and/or to improve efficacy in infecting only a cell type ofinterest,

-   -   and optionally the hybrid AAV is retargeted or engineered as a        hybrid serotype by one or more modifications comprising: 1) a        transcapsidation, 2) adsorption of a bi-specific antibody to a        capsid surface, 3) engineering a mosaic capsid, and/or 4)        engineering a chimeric capsid;    -   (iii) an ApoA-I Binding Protein (APOA1BP, AIBP, or        AI-BP)-inducing compound or composition;    -   (iv) the formulation or pharmaceutical composition of any of (i)        to (iii), wherein the an ApoA-I Binding Protein (APOA1BP, AIBP,        or AI-BP) polypeptide or protein is a human or a mammalian        APOA1BP, or a AIBP1 or a AIBP2, or a recombinant, peptidomimetic        or a synthetic APOA1BP, or a bioisostere of an ApoA-I Binding        Protein (APOA1BP, AIBP, or AI-BP);    -   (v) the formulation or pharmaceutical composition of any of (i)        to (iv), formulated for administration in vivo; or formulated        for enteral or parenteral administration, or for oral,        subcutaneous (SC), intramuscular (IM), intravenous (IV) or        intrathecal (IT) administration,    -   wherein optionally the formulation or pharmaceutical        composition, or the recombinant, peptidomimetic or a synthetic        APOA1BP, or bioisostere of APOA1BP, or nucleic acid encoding the        APOA1BP, or vector having contained therein a nucleic acid        encoding the APOA1BP, is carried in a nanoparticle, a particle,        a micelle or a liposome or lipoplex, a polymersome, a polyplex        or a dendrimer, which optionally can further comprise or express        a cell or CNS penetrating moiety or peptide or a CNS targeting        moiety or peptide; or    -   (vi) the formulation or pharmaceutical composition of any of (i)        to (v), formulated for as a nanoparticle, a liposome, a tablet,        a pill, a capsule, a gel, a geltab, a liquid, a powder, an        emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous        or a sterile or an injectable solution, or an implant (e.g., an        intrathecal implant),

and wherein the formulation or a therapeutic combination is administeredto an individual or patient in need thereof.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes to the same extentas if each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments providedherein and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1 schematically illustrates how AIBP targets Toll-like receptor 4(TLR4)-occupied lipid rafts, facilitates cholesterol efflux and inhibitsTLR4-mediated neuroinflammatory mechanisms of neuropathic pain, asdiscussed in detail in Example 1, below.

FIG. 2 graphically illustrates data showing the role of TLR4 innociceptive processing. WT (n=7) and Tlr4^(−/−) (n=4) mice were treatedwith six i.p. injections of cisplatin (2.3 mg/kg) over a period of 11days (shaded box) and von Frey tested for allodynia. Mean±SEM; *,p<0.05, as discussed in detail in Example 1, below.

FIG. 3 graphically illustrates data showing that AIBP augmentscholesterol efflux from endothelial cells (EC) and macrophages.Mean±SEM; n=4-7; *, p<0.05, as discussed in detail in Example 1, below

FIG. 4A, FIG. 4B and FIG. 4C illustrate images of gels, and FIG. 4Dgraphically illustrates data, showing that: AIBP/HDL₃ inhibitLPS-induced TLR4 dimerization FIG. 4(A); inflammatory signaling inmacrophages in response to LPS FIG. 4(B); TLR4 and NOX4 localization tolipid rafts FIG. 4(C); and, ROS generation FIG. 4(D); in endothelialcells, as discussed in detail in Example 1, below.

FIG. 5 graphically illustrates data showing that intrathecal (i.t.)administration of recombinant AIBP prevents LPS-induced tactileallodynia. Following baseline von Frey threshold testing, mice weregiven an i.t. injection of AIBP (0.5 μg/5 μl), heat-inactivated hi-AIBP(0.5 μg/5 μl), or saline (5 μl). Two hours later, all mice were given ani.t. injection of LPS (0.1 μg/5 μl). Mean±SEM; n=4; *, p<0.05, asdiscussed in detail in Example 1, below.

FIG. 6 graphically illustrates data showing that intrathecaladministration of recombinant AIBP alleviates pre-existing allodynia.Following baseline von Frey threshold testing, animals were given ani.t. LPS (0.1 μg/5 μl). Twenty-four hours later, mice received i.t. AIBP(0.5 μg/5 μl) or saline (5 μl). Mean±SEM; n=4 per group; *, p<0.05, asdiscussed in detail in Example 1, below.

FIG. 7 graphically illustrates data showing that intrathecaladministration of recombinant AIBP prevents intraplantar formalin-evokeddelayed tactile allodynia. Following baseline von Frey thresholdtesting, mice were given an i.t. injection of AIBP (0.5 μg/5 μl) orsaline (5 μl). Two hours later, all mice were given an injection offormalin into one paw, as discussed in detail in Example 1, below.

FIG. 8 graphically illustrates data showing that intrathecaladministration of recombinant AIBP reduces cisplatin-induced tactileallodynia. Mice (n=18) received 6 i.p. injections of cisplatin (2.3mg/kg) over a period of 11 days (gray shaded box). This treatmentresults in a progressive and persistent decrease in tactile threshold.On day 25, twelve mice were given i.t. AIBP (0.5 μg/5 μl; red circles onthe graph) and 6 mice i.t. saline (grey squares). On day 30, the micewere given 2^(nd) i.t. injection: 1) the mice that received saline wereinjected again with saline (grey squares); 2) six mice that receivedAIBP were injected again with AIBP (red circles); and 3) six mice thatreceived AIBP were injected with saline (black circles). y were given asecond i.t. injection of AIBP (n=6) or saline (n=6). Mean±SEM. *,p<0.05, as discussed in detail in Example 1, below.

FIG. 9 graphically illustrates data showing that AIBP i.t. injection andTLR4 deficiency protect against NMDA (N-Methyl-D-aspartic acid orN-Methyl-D-aspartate)-induced allodynia. Two hours after WT micereceived i.t. AIBP (0.5 μg/5 μl; n=8) or no injection (n=8), theyreceived i.t. NMDA (0.25 nM). Mean±SEM; *, p<0.05; **, p<0.005, asdiscussed in detail in Example 1, below.

FIG. 10 illustrates data demonstrating that AIBP binds TLR4 but notTLR1, TLR7 or TLR9; yeast two-hybrid was performed with pLexA-AIBP andTLR ectodomains cloned into pB42AD, as discussed in detail in Example 2,below.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, illustrate that AIBP binds TLR4and inhibits TLR4 dimerization, as discussed in detail in Example 2,below.

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D illustrates that AIBP reducesinflammatory responses in microglia, as discussed in detail in Example2, below.

FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F illustratedata showing that intrathecal AIBP prevents and reverses allodynia, asdiscussed in detail in Example 2, below.

FIG. 14 graphically illustrates data from studies of Intrathecalinjections of AIBP and LPS in female mice, as discussed in detail inExample 2, below.

FIG. 15 graphically illustrates data from studies showing thatIntrathecal AIBP does not affect acute post-formalin paw flinching, asdiscussed in detail in Example 2, below.

FIG. 16A-B schematically and graphically illustrate that AIBP reducesTLR4 occupancy in microglia lipid rafts, as discussed in detail inExample 3, below.

FIG. 17A-B graphically illustrate data showing that AIBP reduces lipidrafts in vivo in spinal cord microglia, as discussed in detail inExample 3, below.

FIG. 18A-D schematically and graphically illustrate that AIBP reducesinflammatory responses in microglia, as discussed in detail in Example3, below.

FIG. 19A-C graphically illustrate data showing that AIBP reducesneuroinflammation in the CNS, as discussed in detail in Example 3,below.

FIG. 20 schematically illustrates an AAV vector used to demonstrate AIBPdelivery to the CNS, where the pAAV-MCS Multiple Cloning Site Region isSEQ ID NO:1; as discussed in detail in Example 4, below.

FIG. 21 illustrates an image of an immunoblot showing mAIBP expressionin HEK293 cells infected with FIB-mAIBP-His-AAV-DJ/8; the blot wasprobed with an anti-His antibody, as discussed in detail in Example 4,below.

FIG. 22 graphically illustrates that intravenous AIBP reduces lightaversion (measure of migraine) in male mice injected with Compound48/80, as discussed in detail in Example 5, below.

FIG. 23 graphically illustrates that intravenous AIBP reduces lightaversion (measure of migraine) in female mice injected with Compound48/80, as discussed in detail in Example 5, below.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodimentsprovided herein, examples of which are illustrated in the accompanyingdrawings. The following detailed description is provided to give thereader a better understanding of certain details of aspects andembodiments of the invention, and should not be interpreted as alimitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, provided are compositions and methods usingpharmaceutical compounds and formulations comprising nucleic acids,polypeptides, and gene and polypeptide delivery vehicles for regulatingor manipulating, including adding, maintaining, enhancing orupregulating, the expression of ApoA-I Binding Protein (APOA1BP, AIBP,or AI-BP), and kits comprising all or some of the components forpracticing these compositions and methods. In alternative embodiments,provided are compositions and methods for delivering therapeutic levelsof AIBP to the body, including the brain and CNS, including use ofdelivery vehicles targeting and/or capable of penetrating the bloodbrain barrier, and nucleic acid (gene) delivery vehicles such as vectorsand viruses such as an adeno-associated virus (AAV) delivery vehiclehaving contained within an AIBP expressing nucleic acid; and for directdelivery of either AIBP polypeptide or AIBP-expressing nucleic aciddirectly via intrathecal (i.t.) administration.

In the course of studies on the role of lipid rafts in cellular receptorprotein trafficking, we unexpectedly discovered that the apoA-I bindingprotein (AIBP) is an important regulatory component, and that elevatingthe neuraxial levels of AIBP regulates the manifestation of post nerveinjury pain states.

In our study of passive K/BxN serum transfer model of arthritis, weinadvertently used C3H/HeJ mice, which are constitutively deficient infunctional Toll-like receptor 4 (TLR4)¹². We unexpectedly found profoundattenuation of the late, but not early phase allodynia (allodynia is theexperience of pain from a non-painful stimulation of the skin, such aslight touch). Similarly, TLR4 knockout had no effect upon baselinemeasures or allodynia observed in the early inflammatory phase (up today 6), but surprisingly prevented persistent allodynia in the latephase. This effect was mimicked by intrathecal (i.t.) injections ofLPS-RS, a TLR4 antagonist, during the day 6-9 interval¹³. Importantly,TLR4 knockout prevented late phase increases in spinal cytokines andactivation of spinal glia and dorsal root ganglia (DRG) ATF3. Notingthat this invention is not limited by any particular mechanism ofaction, TLR4 was demonstrated to be critical in mediating the transitionfrom acute to persistent pain. Similar data have been generatedfollowing nerve injury and treatment with chemotherapeutics in our laband by others¹⁴⁻¹⁸.

TLR4 and other receptors involved in inflammatory signaling localize,constitutively or upon ligand binding, to lipid rafts, which aremembrane microdomains characterized by high content of cholesterol andsphingomyelin¹⁹⁻²². Receptors lacking signaling domains, such as CD14which delivers ligands to TLR4, also reside in lipid rafts, and thedecreased diffusion rates present in lipid rafts support TLR4dimerization, which is an obligatory step in its signalingcascade^(23,24).

In alternative embodiments, provided are compositions and methods fordelivering therapeutically effective amounts of AIBP (either in the formof AIBP polypeptide or AIBP-expressing nucleic acid) to specificallytarget and facilitate cholesterol efflux in TLR4-occupied lipid raftsfor the selective regulation of inflammatory responses, includingneuroinflammation and associated pain and allodynia. Cholesterol is astructural component of any cellular membrane and its content isparticularly high in lipid rafts. Cholesterol removal from lipid raftsdisrupts TLR4 signaling²⁵. Treatment with methyl-β-cyclodextrin (MβCD)is a common method to deplete cholesterol from the plasma membrane incell culture experiments, which does result in inhibition ofTLR4-mediated inflammatory responses^(26,27). Cyclodextrin derivativesare being developed as a promising therapy for the Niemann-Pick type Cdisease, a severe neurodegenerative lysosomal cholesterol storagedisorder²⁸⁻³¹. However, the physiologic mechanism of cholesterol removalfrom the cell involves the membrane and endosomal ATP-binding cassette(ABC) cholesterol transporters ABCA1 and ABCG1 and the extracellularcholesterol acceptors lipid-poor apoA-I and the HDL, whose major proteinis apoA-I³²⁻³⁴. There is a substantial increase in inflammatory geneexpression in response to TLR4 ligands in Abca1^(−/−)Abcg1^(−/−) cells,with less dramatic changes in cells lacking either ABCA1 orABCG1^(35,36). The cholesterol acceptors HDL and lipid-poor apoA-I and anumber of apoA-I mimetic peptides all reduce inflammatory responses aswell³⁷⁻⁴². Yet, the known mechanisms of cholesterol efflux do not implyany spatial or tissue selectivity, and the indiscriminate character ofcellular cholesterol depletion by cyclodextrins or by apoA-I may limittheir clinical applications. Our studies demonstrated that AIBP is aprotein targeting cholesterol efflux specifically to TLR4-occupied lipidrafts for selective regulation of inflammatory responses.

Products of Manufacture, Kits

Also provided are products of manufacture such as implants or pumps,kits and pharmaceuticals for practicing the methods as provided herein.In alternative embodiments, provided are products of manufacture, kitsand/or pharmaceuticals comprising all the components needed to practicea method as provided herein. In alternative embodiments, kits alsocomprise instructions for practicing a method as provided herein,

Formulations and Pharmaceutical Compositions

In alternative embodiments, provided are pharmaceutical formulations orcompositions comprising nucleic acids and polypeptides for practicingmethods and uses as provided herein to regulate neuropathic pain, themethods comprising upregulating the expression of ApoA-I Binding Protein(APOA1BP, AIBP, or AI-BP). In alternative embodiments, provided arepharmaceutical formulations or compositions for use in in vivo, in vitroor ex vivo methods to treat, prevent, reverse and/or ameliorateneuropathic pain. In alternative embodiments, pharmaceuticalcompositions and formulations used to practice methods and uses asprovided herein comprise APOA1BP nucleic acids and polypeptides orresult in an increase in expression or activity of APOA1BP nucleic acidsand polypeptides are administered to an individual in need thereof in anamount sufficient to treat, prevent, reverse and/or ameliorate, e.g., aneuropathic pain, a neurodegenerative disease or condition, optionally achronic or progressive neurodegenerative disease, optionally Alzheimer'sdisease or a Chronic Traumatic Encephalopathy (CTE) or a relatedtauopathy, a traumatic brain injury (TBI), a posttraumatic stressdisorder, a traumatic war neurosis, or a post-traumatic stress syndrome(PTSS). In alternative embodiments, pharmaceutical compositions andformulations used to practice methods and uses as provided hereincomprise APOA1BP nucleic acids and polypeptides or result in an increasein expression or activity of APOA1BP nucleic acids and polypeptides areadministered to an individual in need thereof in an amount sufficient toprevent or decrease the intensity of and/or frequency of e.g., theneuropathic pain or neurodegenerative disease or condition.

In alternative embodiments, the pharmaceutical compositions used topractice methods and uses as provided herein can be administeredparenterally, topically, orally or by local administration, such as byaerosol or transdermally. The pharmaceutical compositions can beformulated in any way and can be administered in a variety of unitdosage forms depending upon the condition or disease and the degree ofillness, the general medical condition of each patient, the resultingpreferred method of administration and the like. Details on techniquesfor formulation and administration are well described in the scientificand patent literature, see, e.g., the latest edition of Remington'sPharmaceutical Sciences, Maack Publishing Co., Easton Pa.(“Remington's”).

For example, in alternative embodiments, these compositions used topractice methods and uses as provided herein are formulated in a buffer,in a saline solution, in a powder, an emulsion, in a vesicle, in aliposome, in a nanoparticle, in a nanolipoparticle and the like. Inalternative embodiments, the compositions can be formulated in any wayand can be applied in a variety of concentrations and forms depending onthe desired in vivo, in vitro or ex vivo conditions, a desired in vivo,in vitro or ex vivo method of administration and the like. Details ontechniques for in vivo, in vitro or ex vivo formulations andadministrations are well described in the scientific and patentliterature. Formulations and/or carriers used to practice methods oruses as provided herein can be in forms such as tablets, pills, powders,capsules, liquids, gels, syrups, slurries, suspensions, etc., suitablefor in vivo, in vitro or ex vivo applications.

In alternative embodiments, formulations and pharmaceutical compositionsused to practice methods and uses as provided herein can comprise asolution of compositions (which include peptidomimetics, racemicmixtures or racemates, isomers, stereoisomers, derivatives and/oranalogs of compounds) disposed in or dissolved in a pharmaceuticallyacceptable carrier, e.g., acceptable vehicles and solvents that can beemployed include water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any fixed oil can be employedincluding synthetic mono- or diglycerides, or fatty acids such as oleicacid. In one embodiment, solutions and formulations used to practicemethods and uses as provided herein are sterile and can be manufacturedto be generally free of undesirable matter. In one embodiment, thesesolutions and formulations are sterilized by conventional, well knownsterilization techniques.

The solutions and formulations used to practice methods and uses asprovided herein can comprise auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and can be selected primarily based on fluid volumes,viscosities and the like, in accordance with the particular mode of invivo, in vitro or ex vivo administration selected and the desiredresults.

The compositions and formulations used to practice methods and uses asprovided herein can be delivered by the use of liposomes. By usingliposomes, particularly where the liposome surface carries ligandsspecific for target cells (e.g., an injured or diseased neuronal cell orCNS tissue), or are otherwise preferentially directed to a specifictissue or organ type, one can focus the delivery of the active agentinto a target cells in an in vivo, in vitro or ex vivo application.

Nanoparticles, Nanolipoparticles and Liposomes

Also provided are nanoparticles, nanolipoparticles, vesicles andliposomal membranes comprising compounds used to practice methods anduses as provided herein, e.g., to deliver compositions comprisingAPOA1BP nucleic acids and polypeptides in vivo, e.g., to the CNS andbrain. In alternative embodiments, these compositions are designed totarget specific molecules, including biologic molecules, such aspolypeptides, including cell surface polypeptides, e.g., for targeting adesired cell type or organ, e.g., a nerve cell or the CNS, and the like.

Provided are multilayered liposomes comprising compounds used topractice methods and uses as provided herein, e.g., as described inPark, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomescan be prepared using a mixture of oil-phase components comprisingsqualane, sterols, ceramides, neutral lipids or oils, fatty acids andlecithins, to about 200 to 5000 nm in particle size, to entrap acomposition used to practice methods and uses as provided herein.

Liposomes can be made using any method, e.g., as described in Park, etal., U.S. Pat. Pub. No. 20070042031, including method of producing aliposome by encapsulating an active agent (e.g., APOA1BP nucleic acidsand polypeptides), the method comprising providing an aqueous solutionin a first reservoir; providing an organic lipid solution in a secondreservoir, and then mixing the aqueous solution with the organic lipidsolution in a first mixing region to produce a liposome solution, wherethe organic lipid solution mixes with the aqueous solution tosubstantially instantaneously produce a liposome encapsulating theactive agent; and immediately then mixing the liposome solution with abuffer solution to produce a diluted liposome solution.

In one embodiment, liposome compositions used to practice methods anduses as provided herein comprise a substituted ammonium and/orpolyanions, e.g., for targeting delivery of a compound (e.g., a APOA1BPnucleic acid and polypeptide) to a desired cell type (e.g., anendothelial cell, a nerve cell, or any tissue or area, e.g., a CNS, inneed thereof), as described e.g., in U.S. Pat. Pub. No. 20070110798.

Provided are nanoparticles comprising compounds (e.g., APOA1BP nucleicacids and polypeptides used to practice methods provided herein) in theform of active agent-containing nanoparticles (e.g., a secondarynanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. Inone embodiment, provided are nanoparticles comprising a fat-solubleactive agent or a fat-solubilized water-soluble active agent to act witha bivalent or trivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate andto deliver compositions used to practice methods and uses as providedherein to mammalian cells in vivo, e.g., to the CNS, as described, e.g.,in U.S. Pat. Pub. No. 20050136121.

Delivery Vehicle Modifications and Modification of AIBP

In alternative embodiments, AIBP peptides or polypeptides, orAIBP-comprising nanoparticles, liposomes and the like (e.g., comprisingor having contained therein APOA1BP nucleic acids or polypeptides usedto practice methods provided herein) are modified to facilitateintrathecal injection, e.g., delivery into the cerebrospinal fluid orbrain. For example, in alternative embodiments, AIBP peptides orpolypeptides, or AIBP-comprising nanoparticles, liposomes and the like,are engineered to comprise a moiety that allows the AIBP peptides orpolypeptides, or AIBP-comprising nanoparticles, liposomes and the like,to bind to a receptor or cell membrane structure that facilitatesdelivery into the CNS or brain, e.g., where the moiety can comprise amannose-6-phosphate receptor, a melanotransferrin receptor, a LRPreceptor or any other receptor that is ubiquitously expressed on thesurface of any CNS or brain cell. For example, conjugation ofmannose-6-phosphate moieties allows the AIBP peptides or polypeptides,or AIBP-comprising nanoparticles, liposomes and the like, to be taken upby a CNS cell that expresses a mannose-6-phosphate receptor. Inalternative embodiments, any protocol or modification of the AIBPpeptides or polypeptides, or AIBP-comprising nanoparticles, liposomesand the like, that facilitates entry or delivery into the CNS or brainin vivo can be used, e.g., as described in U.S. Pat. No. 9,089,566.

In alternative embodiments, AIBP peptides or polypeptides, orAIBP-comprising nanoparticles, liposomes and the like (e.g., comprisingor having contained therein APOA1BP nucleic acids or polypeptides usedto practice methods provided herein) are directly or indirectly linkedor conjugated to any blood brain barrier (BBB)-targeting agent, forexample, a transferrin, an insulin, a leptin, an insulin-like growthfactor, a cationic peptide, a lectin, a Receptor-Associated Protein(RAP) (a 39 kD chaperone localized to the endoplasmic reticulum andGolgi, a lipoprotein receptor-related protein (LRP) receptor familyligand), an antibody (e.g., a peptidomimetic monoclonal antibody) to atransferrin receptor, an antibody (e.g., a peptidomimetic monoclonalantibody) to the insulin receptor, an antibody (e.g., a peptidomimeticmonoclonal antibody) to the insulin-like growth factor receptor, anantibody (e.g., a peptidomimetic monoclonal antibody) to the leptinreceptor and the like. In alternative embodiments, any protocol ormodification of the AIBP peptides or polypeptides, or AIBP-comprisingnanoparticles, liposomes and the like, that facilitates crossing of theBBB can be used, e.g., as described in US Pat App Pub nos. 20050142141;20050042227. For example, to enhance CNS or brain delivery of ancomposition used to practice methods as provided herein, any protocolcan be used, e.g.: direct intra-cranial injection, transientpermeabilization of the BBB, and/or modification of AIBP peptides orpolypeptides, or AIBP-comprising nanoparticles, liposomes and the liketo alter tissue distribution

Delivery Cells and Delivery Vehicles

In alternative embodiments, any delivery vehicle can be used to practicethe methods or uses as provided herein, e.g., to deliver compositions(e.g., APOA1BP nucleic acids and polypeptides) to a CNS or a brain invivo. For example, delivery vehicles comprising polycations, cationicpolymers and/or cationic peptides, such as polyethyleneiminederivatives, can be used e.g. as described, e.g., in U.S. Pat. Pub. No.20060083737. In one embodiment, a delivery vehicle is a transduced cellengineered to express or overexpress and then secrete an endogenous orexogenous AIBP.

In one embodiment, a dried polypeptide-surfactant complex is used toformulate a composition used to practice methods as provided herein,e.g. as described, e.g., in U.S. Pat. Pub. No. 20040151766.

In one embodiment, a composition used to practice methods and uses asprovided herein can be applied to cells using vehicles with cellmembrane-permeant peptide conjugates, e.g., as described in U.S. Pat.Nos. 7,306,783; 6,589,503. In one aspect, the composition to bedelivered is conjugated to a cell membrane-permeant peptide. In oneembodiment, the composition to be delivered and/or the delivery vehicleare conjugated to a transport-mediating peptide, e.g., as described inU.S. Pat. No. 5,846,743, describing transport-mediating peptides thatare highly basic and bind to poly-phosphoinositides.

In one embodiment, cells that will be subsequently delivered to a CNS ora brain are transfected or transduced with an AIBP-expressing nucleicacid, e.g., a vector, e.g., by electro-permeabilization, which can beused as a primary or adjunctive means to deliver the composition to acell, e.g., using any electroporation system as described e.g. in U.S.Pat. Nos. 7,109,034; 6,261,815; 5,874,268.

In Vivo Delivery of AIBP-Encoding Nucleic

In alternative embodiments, provided are compositions and methods fordelivering nucleic acids encoding AIBP peptides or polypeptides, ornucleic acids encoding peptides or polypeptides having AIBP activity, orvectors or recombinant viruses having contained therein these nucleicacids. In alternative embodiments, the nucleic acids, vectors orrecombinant viruses are designed for in vivo or CNS delivery andexpression.

In alternative embodiments, provided are compositions and methods forthe delivery and controlled expression of an AIBP-encoding nucleic acidor gene, or an expression vehicle (e.g., vector, recombinant virus, andthe like) comprising (having contained therein) an AIBP encoding nucleicacid or gene, that results in an AIBP protein being released into thebloodstream or general circulation where it can have a beneficial effecton in the body, e.g., such as the CNS, brain or other targets.

In alternative embodiments, the provided are methods for being able toturn on and turn off AIBP-expressing nucleic acid or gene expressioneasily and efficiently for tailored treatments and insurance of optimalsafety.

In alternative embodiments, AIBP protein or proteins expressed by theAIBP-expressing nucleic acid(s) or gene(s) have a beneficial orfavorable effects (e.g., therapeutic or prophylactic) on a tissue or anorgan, e.g., the brain, CNS, or other targets, even though secreted intothe blood or general circulation at a distance (e.g., anatomicallyremote) from their site or sites of action.

In alternative embodiments, provided are expression vehicles, vectors,recombinant viruses and the like for in vivo expression of anAIBP-encoding nucleic acid or gene to practice the methods as provideherein. In alternative embodiments, the expression vehicles, vectors,recombinant viruses and the like expressing the an AIBP nucleic acid orgene can be delivered by intramuscular (IM) injection, by intravenous(IV) injection, by subcutaneous injection, by inhalation, by a biolisticparticle delivery system (e.g., a so-called “gene gun”), and the like,e.g., as an outpatient, e.g., during an office visit.

In alternative embodiments, this “peripheral” mode of delivery, e.g.,expression vehicles, vectors, recombinant viruses and the like injectedIM or IV, can circumvent problems encountered when genes or nucleicacids are expressed directly in an organ (e.g., the brain or CNS)itself. Sustained secretion of an AIBP in the bloodstream or generalcirculation also circumvents the difficulties and expense ofadministering proteins by infusion.

In alternative embodiments a recombinant virus (e.g., a long-term virusor viral vector), or a vector, or an expression vector, and the like,can be injected, e.g., in a systemic vein (e.g., IV), or byintramuscular (IM) injection, by inhalation, or by a biolistic particledelivery system (e.g., a so-called “gene gun”), e.g., as an outpatient,e.g., in a physician's office. In alternative embodiments, days or weekslater (e.g., four weeks later), the individual, patient or subject isadministered (e.g., inhales, is injected or swallows), a chemical orpharmaceutical that induces expression of the AIBP-expressing nucleicacids or genes; for example, an oral antibiotic (e.g., doxycycline orrapamycin) is administered once daily (or more or less often), whichwill activate the expression of the gene. In alternative embodiments,after the “activation”, or inducement of expression (e.g., by aninducible promoter) of the nucleic acid or gene, an AIBP protein issynthesized and released into the subject's circulation (e.g., into theblood), and subsequently has favorable physiological effects, e.g.,therapeutic or prophylactic, that benefit the individual or patient(e.g., benefit heart, kidney or lung function). When the physician orsubject desires discontinuation of the AIBP treatment, the subjectsimply stops taking the activating chemical or pharmaceutical, e.g.,antibiotic. Alternative embodiments comprise use of “expressioncassettes” comprising or having contained therein a nucleotide sequenceused to practice methods provided herein, e.g., an AIBP-expressingnucleic acid, which can be capable of affecting expression of thenucleic acid, e.g., as a structural gene or a transcript (e.g., encodingan AIBP protein) in a host compatible with such sequences. Expressioncassettes can include at least a promoter operably linked with thepolypeptide coding sequence or inhibitory sequence; and, in one aspect,with other sequences, e.g., transcription termination signals.Additional factors necessary or helpful in effecting expression may alsobe used, e.g., enhancers.

In alternative aspects, expression cassettes also include plasmids,expression vectors, recombinant viruses, any form of recombinant “nakedDNA” vector, and the like. In alternative aspects, a “vector” cancomprise a nucleic acid that can infect, transfect, transiently orpermanently transduce a cell. In alternative aspects, a vector can be anaked nucleic acid, or a nucleic acid complexed with protein or lipid.In alternative aspects, vectors can comprise viral or bacterial nucleicacids and/or proteins, and/or membranes (e.g., a cell membrane, a virallipid envelope, etc.). In alternative aspects, vectors can include, butare not limited to replicons (e.g., RNA replicons, bacteriophages) towhich fragments of DNA may be attached and become replicated. Vectorsthus include, but are not limited to RNA, autonomous self-replicatingcircular or linear DNA or RNA (e.g., plasmids, viruses, and the like,see, e.g., U.S. Pat. No. 5,217,879), and can include both the expressionand non-expression plasmids. In alternative aspects, a vector can bestably replicated by the cells during mitosis as an autonomousstructure, or can be incorporated within the host's genome.

In alternative aspects, “promoters” include all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a mammaliancell such as a muscle, nerve or brain cell. Promoters used in theconstructs provided herein include cis-acting transcriptional controlelements and regulatory sequences that are involved in regulating ormodulating the timing and/or rate of transcription of a nucleic acid,e.g., an AIBP-encoding nucleic acid. For example, a promoter can be acis-acting transcriptional control element, including an enhancer, apromoter, a transcription terminator, an origin of replication, achromosomal integration sequence, 5′ and 3′ untranslated regions, or anintronic sequence, which are involved in transcriptional regulation.These cis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription.

In alternative embodiments, “constitutive” promoters can be those thatdrive expression continuously under most environmental conditions andstates of development or cell differentiation. In alternativeembodiments, “inducible” or “regulatable” promoters can directexpression of a nucleic acid, e.g., an AIBP-encoding nucleic acid, underthe influence of environmental conditions, administered chemical agents,or developmental conditions.

Gene Therapy and Gene Delivery Vehicles

In alternative embodiments, methods of the invention comprise use ofnucleic acid (e.g., gene or polypeptide encoding an AIBP-encodingnucleic acid) delivery systems to deliver a payload of the nucleic acidor gene, or AIBP-expressing nucleic acid, transcript or message, to acell or cells in vitro, ex vivo, or in vivo, e.g., as gene therapydelivery vehicles.

In alternative embodiments, expression vehicle, vector, recombinantvirus, or equivalents used to practice methods provided herein are orcomprise: an adeno-associated virus (AAV), a lentiviral vector or anadenovirus vector; an AAV serotype AAV5, AAV6, AAV8 or AAV9; arhesus-derived AAV, or the rhesus-derived AAV AAVrh.10hCLN2; anorgan-tropic AAV, or a neurotropic AAV; and/or an AAV capsid mutant orAAV hybrid serotype. In alternative embodiments, the AAV is engineeredto increase efficiency in targeting a specific cell type that isnon-permissive to a wild type (wt) AAV and/or to improve efficacy ininfecting only a cell type of interest. In alternative embodiments, thehybrid AAV is retargeted or engineered as a hybrid serotype by one ormore modifications comprising: 1) a transcapsidation, 2) adsorption of abi-specific antibody to a capsid surface, 3) engineering a mosaiccapsid, and/or 4) engineering a chimeric capsid. It is well known in theart how to engineer an adeno-associated virus (AAV) capsid in order toincrease efficiency in targeting specific cell types that arenon-permissive to wild type (wt) viruses and to improve efficacy ininfecting only the cell type of interest; see e.g., Wu et al., Mol.Ther. 2006 September; 14(3):316-27. Epub 2006 July 7; Choi, et al.,Curr. Gene Ther. 2005 June; 5(3):299-310.

For example, in alternative embodiments, serotypes AAV-8, AAV-9, AAV-DJor AAV-DJ/8™ (Cell Biolabs, Inc., San Diego, Calif.), which haveincreased uptake in brain tissue in vivo, are used to deliver anAIBP-encoding nucleic acid payload for expression in the CNS. Inalternative embodiments, the following serotypes, or variants thereof,are used for targeting a specific tissue:

Tissue Optimal Serotype CNS AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 HeartAAV1, AAV8, AAV9 Kidney AAV2 Liver AAV7, AAV8, AAV9 Lung AAV4, AAV5,AAV6, AAV9 Pancreas AAV8 Photoreceptor Cells AAV2, AAV5, AAV8 RPE(Retinal AAV1, AAV2, AAV4, AAV5, AAV8 Pigment Epithelium) SkeletalMuscle AAV1, AAV6, AAV7, AAV8, AAV9

In alternative embodiments, the rhesus-derived AAV AAVrh.10hCLN2 orequivalents thereof can be used, wherein the rhesus-derived AAV may notbe inhibited by any pre-existing immunity in a human; see e.g., Sondhi,et al., Hum Gene Ther. Methods. 2012 October; 23(5):324-35, Epub 2012Nov. 6; Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct. 17; teachingthat direct administration of AAVrh.10hCLN2 to the CNS of rats andnon-human primates at doses scalable to humans has an acceptable safetyprofile and mediates significant payload expression in the CNS.

Because adeno-associated viruses (AAVs) are common infective agents ofprimates, and as such, healthy primates carry a large pool ofAAV-specific neutralizing antibodies (NAbs) which inhibit AAV-mediatedgene transfer therapeutic strategies, methods provided herein cancomprise screening of patient candidates for AAV-specific NAbs prior totreatment, especially with the frequently used AAV8 capsid component, tofacilitate individualized treatment design and enhance therapeuticefficacy; see, e.g., Sun, et al., J. Immunol. Methods. 2013 Jan. 31;387(1-2):114-20, Epub 2012 Oct. 11.

Dosaging

The pharmaceutical compositions and formulations used to practicemethods and uses as provided herein can be administered for prophylacticand/or therapeutic treatments. In therapeutic applications, compositionsare administered to a subject already suffering from a disease,condition, infection or defect in an amount sufficient to cure,alleviate or partially arrest the clinical manifestations of thedisease, condition, infection or disease and its complications (a“therapeutically effective amount”), including e.g., a neuropathic pain.For example, in alternative embodiments, APOA1BP nucleic acid- orpolypeptide-comprising pharmaceutical compositions and formulations asprovided herein are administered to an individual in need thereof in anamount sufficient to treat, prevent, reverse and/or ameliorate aneuropathic pain, an inflammation-induced neuropathic pain, aninflammation-induced neuropathic pain, a nerve or CNS inflammation, aallodynia, a post nerve injury pain or neuropathic pain, a post-surgicalpain or neuropathic pain, a chemotherapeutic-induced peripheralneuropathy (CIPN) (e.g., cisplatin-induced allodynia), aneurodegenerative disease or condition, optionally a chronic orprogressive neurodegenerative disease or condition, optionallyAlzheimer's disease or a Chronic Traumatic Encephalopathy (CTE) or arelated tauopathy, a traumatic brain injury (TBI), a posttraumaticstress disorder, a traumatic war neurosis, or a post-traumatic stresssyndrome (PTSS), a migraine, and/or a hyperalgesia.

The amount of pharmaceutical composition adequate to accomplish this isdefined as a “therapeutically effective dose.” The dosage schedule andamounts effective for this use, i.e., the “dosing regimen,” will dependupon a variety of factors, including the stage of the disease orcondition, the severity of the disease or condition, the general stateof the patient's health, the patient's physical status, age and thelike. In calculating the dosage regimen for a patient, the mode ofadministration also is taken into consideration.

In alternative embodiments, viral vectors such as adenovirus or AAVvectors are administered to an individual in need therein, and inalternative embodiment the dosage administered to a human comprises: adose of about 2×10¹² vector genomes per kg body weight (vg/kg), orbetween about 10¹⁰ and 10¹⁴ vector genomes per kg body weight (vg/kg),or about 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or more vg/kg, whichcan be administered as a single dosage or in multiple dosages, asneeded. In alternative embodiments, these dosages are administeredorally, IM, IV, or intrathecally. In alternative embodiments, thevectors are delivered as formulations or pharmaceutical preparations,e.g., where the vectors are contained in a nanoparticle, a particle, amicelle or a liposome or lipoplex, a polymersome, a polyplex or adendrimer. In alternative embodiments, these dosages are administeredonce a day, once a week, or any variation thereof as needed to maintainin vivo expression levels of AIBP, which can be monitored by measuringactually expression of AIBP or by monitoring of therapeutic effect,e.g., diminishing of pain. The dosage regimen also takes intoconsideration pharmacokinetics parameters well known in the art, i.e.,the active agents' rate of absorption, bioavailability, metabolism,clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. SteroidBiochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341;Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci.84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur.J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra). Thestate of the art allows the clinician to determine the dosage regimenfor each individual patient, active agent and disease or conditiontreated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods as provided herein are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on the dosage and frequency as required and tolerated by thepatient. The formulations should provide a sufficient quantity of activeagent to effectively treat, prevent or ameliorate a conditions, diseasesor symptoms as described herein. For example, alternative exemplarypharmaceutical formulations for oral administration of compositions usedto practice methods as provided herein are in a daily amount of betweenabout 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram ofbody weight per day. In an alternative embodiment, dosages are fromabout 1 mg to about 4 mg per kg of body weight per patient per day areused. Lower dosages can be used, in contrast to administration orally,into the blood stream, into a body cavity or into a lumen of an organ.Substantially higher dosages can be used in topical or oraladministration or administering by powders, spray or inhalation. Actualmethods for preparing parenterally or non-parenterally administrableformulations will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington's, supra.

The methods as provided herein can further comprise co-administrationwith other drugs or pharmaceuticals, e.g., compositions for treating anyneurological or neuromuscular disease, condition, infection or injury,including related inflammatory and autoimmune diseases and conditions,and the like. For example, the methods and/or compositions andformulations as provided herein can be co-formulated with and/orco-administered with, fluids, antibiotics, cytokines, immunoregulatoryagents, anti-inflammatory agents, pain alleviating compounds, complementactivating agents, such as peptides or proteins comprising collagen-likedomains or fibrinogen-like domains (e.g., a ficolin),carbohydrate-binding domains, and the like and combinations thereof

Bioisosteres of Compounds

In alternative embodiment, also provided are bioisosteres of compoundsused to practice the methods provided herein, e.g., polypeptides havinga APOA1BP activity. Bioisosteres used to practice methods as providedherein include bioisosteres of, e.g., APOA1BP nucleic acids andpolypeptides, which in alternative embodiments can comprise one or moresubstituent and/or group replacements with a substituent and/or grouphaving substantially similar physical or chemical properties whichproduce substantially similar biological properties to compounds used topractice methods or uses as provided herein. In one embodiment, thepurpose of exchanging one bioisostere for another is to enhance thedesired biological or physical properties of a compound without makingsignificant changes in chemical structures.

For example, in one embodiment, one or more hydrogen atom(s) is replacedwith one or more fluorine atom(s), e.g., at a site of metabolicoxidation; this may prevent metabolism (catabolism) from taking place.Because the fluorine atom is similar in size to the hydrogen atom theoverall topology of the molecule is not significantly affected, leavingthe desired biological activity unaffected. However, with a blockedpathway for metabolism, the molecule may have a longer half-life or beless toxic, and the like.

Devices for Delivering Therapeutic Agents Directly into the CNS or Brain

In alternative embodiments, pharmaceutical compositions andformulations, including nanoparticles and liposomes, used to practicemethods as provided herein are delivered directly into a CNS or a brain,e.g., either by injection intravenously or intrathecally, or by variousdevices known in the art. For example, U.S. Pat. App. Pub. No.20080140056, describes a rostrally advancing catheter in the intrathecalspace for direct brain delivery of pharmaceuticals and formulations.Implantable infusion devices can also be used; e.g., a catheter todeliver fluid from the infusion device to the brain can be tunneledsubcutaneously from the abdomen to the patient's skull, where thecatheter can gain access to the individual's brain via a drilled hole.Alternatively, a catheter may be implanted such that it delivers theagent intrathecally within the patient's spinal canal. Flexible guidecatheters having a distal end for introduction beneath the skull of apatient and a proximal end remaining external of the patient also can beused, e.g., see U.S. Pat. App. Pub. No. 20060129126.

In alternative embodiments, pharmaceutical compositions and formulationsused to practice methods as provided herein are delivered via directdelivery of pharmaceutical compositions and formulations, includingnanoparticles and liposomes, or direct implantation of cells thatexpress AIBP into a brain, for example, using any cell implantationcannula, syringe and the like, as described e.g., in U.S. Pat. App. Pub.No. 20080132878; or elongate medical insertion devices as describede.g., in U.S. Pat. No. 7,343,205; or a surgical cannula as describede.g., in U.S. Pat. No. 4,899,729. Implantation cannulas, syringes andthe like also can be used for direct injection of liquids, e.g., asfluid suspensions.

In alternative embodiments, pharmaceutical compositions and formulationsused to practice methods as provided herein are delivered with tracersthat are detectable, for example, by magnetic resonance imaging (MRI)and/or by X-ray computed tomography (CT); the tracers can be co-infusedwith the therapeutic agent and used to monitor the distribution of thetherapeutic agent as it moves through the target tissue, as describede.g., in U.S. Pat. No. 7,371,225.

Kits and Instructions

Provided are kits comprising compositions (including the devices asdescribed herein) and/or instructions for practicing methods as providedherein to e.g., treat, ameliorate or prevent a neuropathic pain. Assuch, kits, cells, vectors and the like can also be provided. Inalternative embodiments, provided are kits comprising: a compositionused to practice a method as provided herein, or a composition, apharmaceutical composition or a formulation as provided herein, andoptionally comprising instructions for use thereof.

The invention will be further described with reference to the examplesdescribed herein; however, it is to be understood that the invention isnot limited to such examples.

EXAMPLES Example 1: Efficacy Demonstrated in Exemplary Methods

This example describes and demonstrates exemplary embodiments, and theefficacy of methods as provided herein to e.g., treat or ameliorate aneuropathic pain, including e.g., allodynia and TLR4-mediatedinflammation-induced neuropathic pain.

Role of TLR4 in Nociceptive Processing.

TLR4 serves as a pervasive mediator of persistent pain states. FIG. 2shows that TLR4 deficiency results in a complete attenuation of tactileallodynia after treatment with the chemotherapeutic cisplatin²⁰. Boththe initial development of allodynia, during cisplatin treatment, andthe persistent pain state are attenuated. We have shown thatTLR4-mediated release of TNFα is considered to be an important factor inthe development of allodynia^(16,59,60).

TLR4 localizes to lipid rafts, and the factors that facilitatecholesterol depletion from lipid rafts have been shown to diminishTLR4-mediated inflammatory responses^(20,61-64.) Below we describe anovel player in regulation of lipid rafts, AIBP, which we demonstratecan modulate (attenuate) TLR4-mediated allodynia and hyperalgesia.

AIBP Augments Cholesterol Efflux, Reduces Lipid Rafts and Interfereswith Receptor Signaling.

Since AIBP binds apoA-I⁵³, first we validated that recombinant AIBPbound HDL₃, a major HDL fraction involved in cholesterol efflux⁵⁸. Then,we demonstrated that in the presence of AIBP, cholesterol efflux fromendothelial cells (EC) and from macrophages to HDL₃ was increased 2-4fold (FIG. 3). By itself, AIBP did not promote cholesterol efflux in theabsence of HDL₃. Rather, AIBP binding to cells increased the overallcapacity of the cell to bind HDL₃ and increased the rate of HDL₃dissociation from cells⁵⁸, thereby creating conditions that wouldfacilitate cholesterol efflux.

HDL removes cholesterol and reduces lipid rafts, a process whichinterferes with ligand-induced TLR4 dimerization. This hypothesisreceived initial validation in the experiments shown in FIG. 4:AIBP/HDL₃ treatment inhibited LPS-induced TLR4 dimerization in BaF3cells expressing TLR4-flag and TLR4-gfp (A), LPS-induced phosphorylationof p65 and ERK1/2 in RAW macrophages (B), and localization of TLR4 andNOX4 to lipid rafts in EC (C), with a subsequent reduction inLPS-induced ROS (D).

To test the hypothesis that AIBP inhibits TLR4-mediated inflammatorymechanisms of neuropathic pain, we injected saline or recombinant AIBPintrathecally (i.t.) 2 hours prior to the i.t. injection of LPS, thespecific TLR4 agonist (FIG. 5). As expected from our previous studies,in the saline-injected group, LPS induced severe tactile allodynia.Remarkably, the AIBP injection entirely prevented LPS-induced allodynia,whereas injections of denatured, heat-inactivated AIBP did not have anyeffect. We did not observe any apparent toxicity of i.t. (or i.v. inother experiments) AIBP. These initial results provide strong support toour hypothesis. Importantly, i.t. AIBP injected up to 24 hours afteri.t. LPS also reduced allodynia (FIG. 6).

Intraplantar Formalin-Evoked Delayed Tactile Allodynia.

Intraplantar injection of formalin yields acute flinching behavior and,after 7 days, a persistent tactile allodynia and associated spinalmicroglial activation⁶⁵. In recent work, we found that TLR4 knockout orantagonism had no effect on flinching, but prevented delayed tactileallodynia. Similarly, i.t. injections of AIBP prevented ipsilateral butnot contralateral paw delayed tactile allodynia in response to formalin(FIG. 7).

Cisplatin-Induced Polyneuropathy.

In rodents, as people, cisplatin induces long-lasting tactile allodynia.This highly translational model showed that cisplatin-induced tactileallodynia is attenuated in Tlr4^(−/−) mice^(20,21). We found that asingle i.t. AIBP injection significantly, although transiently (days),reversed established cisplatin-induced allodynia (FIG. 8), while asecond injection produced longer efficacy.

Clinically, chemotherapeutic-induced peripheral neuropathy (CIPN) is oneof the only neuropathic pain states in which the initiation of thepainful stimulus is predictably controlled. Therefore, we examinedwhether i.t. AIBP can prevent, as well as reverse, cisplatin-inducedtactile allodynia in mice. Data graphically illustrated in FIG. 8, asingle dose of AIBP may not be adequate; but as shown in the K/BxNmodel, 3 i.t. injections of a TLR4 antagonist were necessary to promotereversal of the delayed, persistent allodynia¹⁶. We note that multiplei.t. dosing can be performed in mice.

While the invention is not limited by any particular mechanism ofaction, AIBP may affect not only TLR4 but also the function of otherreceptors localized to lipid rafts. We injected mice i.t. with AIBP,followed by i.t. NMDA. Remarkably, the AIBP injection in large partprevented NMDA-induced allodynia (FIG. 9).

Example 2: Efficacy Demonstrated in Exemplary Methods—AIBP Treats andPrevents Neuropathic Pain

This example describes and demonstrates exemplary embodiments, and theefficacy of methods as provided herein to e.g., treat or ameliorate aneuropathic pain, including e.g., allodynia and TLR4-mediatedinflammation-induced neuropathic pain.

In alternative embodiments, provided are methods for treating,ameliorating, preventing, reversing or decreasing the severity orduration of nerve and tissue injury and related neuropathic pain statesgenerated by e.g., trauma, chemotherapy, arthritis, diabetes, or viralinfection, all of which can result in neuropathic pain states.

A common feature of these initiating events is the release ofdamage-associated molecular pattern molecules, which can activateToll-like receptors (TLRs) (1-4). Neuraxial TLR4 and its downstreamsignaling adaptors have been implicated in the development offacilitated pain states, such as occurs after nerve injury (5-11) andparticularly in the evolution of a chronic pain state after an acuteinjury or inflammation (6). As several other key inflammatory andneurotransmitter receptors, activated TLR4 localizes to lipid rafts(12-15). We found that AIBP, in combination with high-densitylipoprotein (HDL), reduces lipid rafts in endothelial cells and therebycontrols activation of lipid raft-localized vascular endothelial growthfactor receptor-2 and restricts angiogenesis (17).

While the invention is not limited by any particular mechanism ofaction, provided are methods for using or administering AIBP to regulateinflammatory receptors residing in lipid rafts, including TLRs inmacrophages and microglia.

Materials and Methods

Animals: All experiments were conducted according to protocols approvedby the Institutional Animal Care and Use Committee of the University ofCalifornia, San Diego. Mice were housed up to 4 per standard cage atroom temperature and maintained on a 12:12 hour light:dark cycle, withlights on at 07:00. All behavioral testing was performed during thelight cycle. Both food and water were available ad libitum. Wild typeC57Bl/6 male mice were purchased from Harlan (Indianapolis, Ind.) or theJackson Lab (Bar Harbor, Me.).

Cells: Primary peritoneal macrophages were harvested from C57Bl/6 mice 3days following an intraperitoneal injection of thioglycollate, selectedby adsorption to a plate and maintained in DMEM supplemented with 10%heat-inactivated FBS (Omega Scientific) and 50 μg/ml gentamicin (OmegaScientific). Primary microglia were isolated from 2 to 3 week oldC57BI/6 mice as previously described (1). In brief, brains isolated from5 to 6 mice were pooled together and homogenized in HBSS (Gibco)supplemented with 0.5% BSA and 1 mM EDTA on ice. A single cellsuspension was obtained with a 70 μm cell strainer (Biologix Group)separated on a discontinuous 37-70% Percoll (GE Healthcare) gradient.After removal of a myelin/debris layer, cells were collected from theinterphase, washed and plated in DMEM/F12 (Cellgro) supplemented with 5%FBS and 50 μg/ml gentamicin. Cells were supplemented daily for 7 dayswith 20 ng/ml of recombinant mouse IL-34 (R&D Systems). Immortalizedmicroglial cell line BV-2 (2) were maintained in DMEM supplemented with5% FBS (Omega Scientific) and 50 μg/ml gentamicin. Ba/F3 cells stablyexpressing TLR4-gfp, TLR4-flag and MD2 (3, 4) were cultured in RPMI1640(Invitrogen) containing 70 units/ml recombinant murine interleukin-3,10% heat-inactivated FBS, 50 μg/ml gentamicin. HEK293 cells werecultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin.

Yeast two-hybrid system: Interactions of the ectodomains of TLRs withAIBP were assessed by a yeast two-hybrid assay (BD Clontech, Palo Alto,Calif.). In one experiment (FIG. 1), the EGY48 yeast transformed withp80p-LacZ were co-transformed with pB42AD-AIBP and pLexA-TLR4 (AA1-629). In a different experiment (FIG. 10), the vectors were switchedand yeast were co-transformed pLexA-AIBP and either pB42AD-TLR1 (AA22-524), pB42AD-TLR4 (AA 1-629), pB42AD-TLR7 (AA 1-795), or pB42AD-TLR9(AA 1-772). Following selection for a Trp⁺ and His⁺ phenotype,Leu-dependent growth and β-gal (LacZ) activity were tested in inductionmedium (SD/galactose/raffinose). The positive control was the yeast cellline EGY48/p80p-LacZ co-transfected with pLexA53 and pB42ADT; thenegative control was the yeast cell line co-transfected with pLexA andpB42AD (5).

AIBP-TLR4 pull-down assay: HEK293 cells were transfected with both theextracellular domain of human flag-TLR4 and human flag-AIBP usingGenJet™ In Vitro DNA transfection reagent (SignaGen Laboratories). At 2days after transfection, cells were lysed with an ice-cold lysis buffer(50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5mM Na₃VO₄, 1 mM NaF, and a protease inhibitor cocktail from Sigma). Celllysates were preincubated with protein A/G sepharose beads (GEHealthcare) for 30 min at 4° C. and immunoprecipitated with a rabbitanti-AIBP antibody (Abcam) overnight at 4° C. Next day, the lysates werefurther incubated with protein A/G Sepharose beads for additional 1 hourat 4° C. Immune complexes were washed five times with lysis buffer, runon a NuPAGE™ gel (Invitrogen), and AIBP-bound TLR4 was detected byimmunoblotting with an anti-flag antibody (Sigma).

Flow cytometry binding assay: Primary peritoneal macrophages isolatedfrom wild type and Tlr4^(−/−) mice were plated overnight, then washedwith PBS, blocked with TBS containing 1% BSA for 30 min on ice, andincubated with either 2 μg/ml BSA or 2 μg/ml AIBP for 2 hours on ice.Cells were washed three times with PBS and incubated with 1 μg/mlFITC-conjugated anti-His antibody (Abcam) for 1 hour at 4° C. After fivewashes with PBS, cells were analyzed using a FACSCanto II™ (BDBiosciences, San Jose, Calif.) flow cytometer. Geometric means of FACShistograms were measured and presented as bar graphs.

TLR4 dimerization assay: Ba/F3 cells were pretreated with either 50μg/ml HDL alone or 50 μg/ml HDL+0.2 μg/ml AIBP. Two hours afterpretreatment, cells were stimulated with either DMSO (vehicle) or 10ng/ml of LPS for 10 min at 37° C. and lysed with an ice-cold lysisbuffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mMEGTA, 5 mM Na₃VO₄, 1 mM NaF, and a protease inhibitor cocktail fromSigma). Cell lysates were preincubated with protein A/G sepharose beadsfor 30 min at 4° C. and immunoprecipitated with a rabbit anti-GFPantibody (Abcam) overnight at 4° C. Next day, the lysates were incubatedwith protein A/G sepharose beads for 1 hour at 4° C. Immune complexeswere washed five times with lysis buffer, run on a NuPAGE™ gel(Invitrogen), and dimerized TLR4 was detected by immunoblotting withanti-flag (Sigma) and anti-GFP antibodies.

Isolation of lipid rafts: Lipid rafts were isolated using adetergent-free, discontinuous gradient ultracentrifugation method as inour earlier work (6). In brief, BV-2 cells were pretreated with eitherBSA or AIBP for 2 hours and then stimulated with 10 ng/ml LPS for 10min. Cells were washed two times with ice-cold PBS and harvested into 1ml of 0.5 M sodium bicarbonate buffer (pH 11.0) containing a proteaseinhibitor cocktail, homogenized and sonicated. One ml of disrupted cellsuspension was mixed with 1 ml of 90% sucrose in MBS (25 mM2-(N-morpholino)ethanesulfonic acid, pH6.5, 150 mM NaCl) to adjust to45% sucrose and placed into ultracentrifugation tubes. Four ml of 5-35%discontinuous sucrose gradient in MBS containing 250 mM sodiumbicarbonate was formed above the sample. Following ultracentrifugationat 35,000 rpm in a SW-41 rotor (Beckman) for 20 hours at 4° C., ten 1 mlfractions were collected from top to bottom. The lipid raft fractions 4and 5 were combined, supplemented with 3× volume of MBS buffer andsubjected to an additional round of ultracentrifugation at 35,000 rpmfor 2 hours at 4° C. The pellet was re-suspended in LDS sample buffer(Invitrogen), and samples were run on a NuPAGE™ gel, transferred to aPVDF membrane and immunoblotted with the indicated antibodies.

Ex vivo flow cytometry for lipid rafts: C57Bl/6 mice were intrathecally(i.t.) injected with saline or AIBP. Two hours later, mice wereintrathecally injected with LPS. Fifteen min after LPS injection, spinalcords were harvested and single-cell suspensions were obtained fromspinal cords using a Neural Tissue Dissociation™ kit (Miltenyi Biotec)according to the manufacturer's protocol. To remove myelin, MyelinRemoval Beads II™ (Miltenyi Biotec) were added to samples and incubatedfor 15 min at 4° C., followed by washes and separation with an LS columnand a MACS™ Separator (Miltenyi Biotec). Following isolation, cells werewashed two times with a FACS buffer (BD Bioscience), incubated with ananti-CD16/CD32 antibody (FcRγ blocker, BD Bioscience) for 10 min at roomtemperature, followed by staining with an APC-conjugated CD11b antibody(BD Bioscience) and FITC-conjugated cholera toxin B (Sigma) for 1 houron ice. Cells were washed two times with a FACS buffer, fixed with 3.7%formaldehyde for 15 min on ice, washed three times with a FACS buffer,and analyzed using a FACSCanto II™ (BD Biosciences) flow cytometer.

Immunoblot. Antibodies specific to FLOT1, phospho-p65, p65,phospho-ERK1/2, ERK1/2, and GAPDH (Cell Signaling Technology) and a TLR4antibody (GenWay Biotech). Antibodies specific to FLAG and GFP werepurchased from Sigma-Aldrich and Abcam, respectively. Cell lysates weresubjected to gel electrophoresis and immunoblot as described(7).

Quantitative PCR. Total RNA was isolated using Nucleospin™ RNA columns(Clontech). Isolated RNA was reverse transcribed using RNA to cDNAEcoDry (Clontech) following the manufacturer's protocol. QuantitativePCR was performed using KAPA SYBR FAST™ Universal qPCR kit (KAPABiosystems, KK4602), with the primers ordered from Integrated DNATechnologies (IDT), and a Rotor Gene Q™ thermocycler (Qiagen).

Recombinant AIBP: AIBP was produced in a baculovirus/insect cell systemto allow for posttranslational modification and to ensure endotoxin-freepreparation. Human AIBP was cloned into a pAcHLT-C vector behind thepolyhedrin promoter. The vector contains an N-terminal His-tag to enablepurification and detection. Insect Sf9 cells were transfected with BDBaculoGold™ Baculovirus DNA and the AIBP vector. After 4-5 days, thesupernatant was collected to afford a baculovirus stock. Fresh Sf9 cellswere infected with the AIBP producing baculovirus, cell pellets werecollected after 3 days, lysed, sonicated, cleared by centrifugation, andthe supernatants loaded onto a Ni-NTA agarose column eluted withimidazole. Protein was dialyzed against saline, and concentrationmeasured. Aliquots were stored at −80° C.

LPS and cyclodextrin: In vitro experiments were conducted withKdo2-LipidA (KLA; Avanti Polar Lipids), a well-characterized activecomponent of LPS and a highly specific TLR4 agonist (8). Our earlierstudies have demonstrated that i.t. injections of KLA or ultra-pure LPSfrom Escherichia coli 0111:B4 (Invivogen) produced identical allodyniaresponses in mice (9). In this study we used for i.t. injectionsInvivogen's LPS at 0.1 μg/5 μl in 0.9% saline. The pharmaceutical gradebeta-cyclodextrin CAVAMAX W7 PHARMA™ was from Wacker Chemie AG.

Cisplatin treatment: Mice received intraperitoneal (i.p.) injections ofcisplatin (2.3 mg/kg/injection; Spectrum Chemical MFG) every other dayfor 6 total injections to induce tactile allodynia. Between cisplatininjection days, lactated Ringer's solution (0.25 ml) was injected tomaintain hydration and to protect the kidney and liver. During theperiod of cisplatin administration, gross behavioral observations weremade and animals were assessed for general health, including changes inbody weight. In case of dehydration, additional lactated Ringer'ssolution was administered. In the study, the criteria for euthanasia wasweight loss in excess of 20%, however, no animals required euthanasia.

Mouse intrathecal (i.t.) injection: Mice were anesthetized using 4%isoflurane for induction and 2.5% for maintenance of anesthesia with amixture of 50% oxygen and 50% room air. The lower back was shaven andthe animal was placed in a prone posture so that the pelvis could beheld between the thumb and forefinger. The L5 and L6 vertebrae wereidentified by palpation and a 30G needle was inserted percutaneously onmidline between the L5 and L6 vertebrae. Successful entry was assessedby the observation of a brisk tail flick. Injections of 5 μl wereadministered over an interval of approximately 30 seconds. Followingrecovery from anesthesia, mice were evaluated for normal motorcoordination and muscle tone.

Mechanical allodynia: For testing, animals were placed in clear,plastic, wire mesh-bottomed cages for 45 min prior to the initiation oftesting. Tactile thresholds were measured with a series of von Freyfilaments (Semmes Weinstein von Frey Anesthesiometer; Stoelting Co.)ranging from 2.44 to 4.31 (0.04-2.00 g). The 50% probability ofwithdrawal threshold was recorded. In light of reports of the possiblecontribution of sex of the experimenter (10), we note that a femaleperformed the mouse behavioral testing. In the present experiments,mechanical withdrawal thresholds were assessed prior to treatment and atenter times post-treatment using the up-down method (11).

Formalin Flinching: A metal band was placed around the left hindpaw ofthe mouse. After 1 hour acclimation with the metal band, the mousereceived a single injection of intraplantar formalin (2.5%) to induceflinching. The movement of the metal band (mouse flinching) was detectedby an automated device (12) for a period of 1 hour after delivery offormalin.

Statistical Analyses: Results were analyzed using Student's t-test (fordifferences between 2 groups) or two-way ANOVA with the Bonferroni posthoc test (for time course experiments), using GraphPad Prism™.Differences between groups with p<0.05 were considered statisticallysignificant. We did not use statistical methods to predetermine samplesize, there was no randomization designed in the experiments, and thestudies were not blinded. Samples sizes were estimated on the basis ofprevious experimental studies. No exclusion criteria were used in thesestudies.

Results and Discussion

Using yeast two-hybrid system, we demonstrated constitutive AIBP bindingto the TLR4 ectodomain, but not to ectodomains of TLR1, TLR7 or TLR9(FIG. 11A and FIG. 10). The AIBP-TLR4 binding was confirmed in apull-down experiment with AIBP and TLR4 ectodomain expressed in HEK 293cells (FIG. 11B). In addition, recombinant AIBP bound to peritonealmacrophages from wild type (WT) but not Tlr4^(−/−) mice (FIG. 11C).These results led us to hypothesize that AIBP targets TLR4-occupiedlipid rafts and that, in turn, AIBP-mediated raft disruption interfereswith ligand-induced TLR4 dimerization. Indeed, AIBP treatment inhibitedLPS-induced TLR4 dimerization in Ba/F3 cells expressing TLR4-flag,TLR4-gfp and MD2 (FIG. 11D).

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, illustrate that AIBP binds TLR4and inhibits TLR4 dimerization: FIG. 11A shows a Yeast two-hybrid studyperformed with pB42AD-AIBP and pLexA-TLR4 ectodomain; FIG. 11Bgraphically illustrates data where HEK293 cells were co-transfected withthe flag-tagged TLR4 ectodomain and flag-tagged AIBP; AIBP from celllysates were pulled downed with an anti-AIBP antibody or an isotypecontrol IgG, and blots of the pull-down or total cell lysates wereprobed with an anti-flag antibody; FIG. 11C graphically illustrates datawhere peritoneal elicited macrophages from WT and Tlr4−/− mice wereincubated for 2 hours on ice with 2 μg/ml BSA or 2 μg/ml AIBP (with aHis-tag) and then subjected to a flow cytometry analysis with aFITC-conjugated anti-His antibody; FIG. 11D graphically illustrates datawhere Ba/F3 cells stably expressing TLR4-gfp, TLR4-flag and MD2 wereincubated with serum-free media containing 50 m/ml HDL, in the presenceor absence of 0.2 μg/ml AIBP, and then stimulated with 10 ng/ml LPS for20 min; cell lysates were immune-precipitated with an anti-GFP antibodyand blots were probed with anti-flag and anti-GFP antibodies; Mean±SEM;n=4-6; **, p<0.01; Student's t-test.

Earlier studies have demonstrated that TLR4 serves as a pervasivemediator of persistent pain states. TLR4 deficiency results in acomplete attenuation of tactile allodynia during treatment with thechemotherapeutic cisplatin and during the persistent pain state afterthe cisplatin treatment was completed (10, 11). Experiments with nerveinjury and arthritis models also support the role of TLR4 in mediatingthe transition from an acute to a persistent pain state (7-11, 18, 19).Tellingly, intrathecal (i.t.) injections of LPS, a specific TLR4 ligand,but not of LPS-RS, which does not activate TLR4, result in immediatetactile allodynia (19). The mechanism involves TLR4-mediated release ofinflammatory cytokines from microglia and/or astrocytes which in turnleads to central sensitization and allodynia (2, 19, 20).

Hence, we tested whether AIBP affects TLR4 in microglia. Exposure to LPSresults in a greater TLR4 recruitment to lipid rafts, where thereceptors dimerize and initiate inflammatory signaling (21). Treatmentwith AIBP reduced LPS-induced TLR4 occupancy in lipid rafts inmicroglia-like BV-2 cells (FIG. 12A). In these cells, AIBP treatmentalso resulted in inhibition of p65 and ERK1/2 phosphorylation (FIG. 12B)and inflammatory cytokine mRNA expression (FIG. 12C) in response to LPS.These results were replicated in primary mouse microglia in which AIBPcompletely inhibited LPS-induced expression of the majority ofinflammatory cytokines (FIG. 12D).

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D illustrate that AIBP reducesinflammatory responses in microglia: FIG. 12A-C illustrate data fromstudies where BV-2 cells were incubated for 2 hours with vehicle (0.1%BSA) or 0.2 μg/ml AIBP (in 0.1% BSA) in serum-containing medium andstimulated with 10 ng/ml LPS. TLR4 occupancy in lipid rafts was tested15 min after adding LPS (FIG. 12A), p65 and ERK1/2 phosphorylation after30 min (FIG. 12B), and cytokine mRNA expression after 2 h of incubation(FIG. 12C); and FIG. 12D graphically illustrates data from studies wherePrimary mouse microglia (pooled from 5-6 mice per sample) were incubatedfor 2 hours with vehicle (0.1% BSA) or 0.2 μg/ml AIBP (in 0.1% BSA) inserum-containing medium and stimulated with 10 ng/ml LPS for 1 hour;Mean±SEM; n=4-6 for BV-2; n=3 for primary microglia experiments; *,p<0.05; **, p<0.01; ****, p<0.0005 (Student's t-test).

To examine whether AIBP reduces lipid rafts in spinal cord microglia invivo, we i.t. injected mice with saline or recombinant AIBP two hoursprior to the i.t. injection of LPS. AIBP significantly reduced theabundance of lipid rafts in spinal microglia compared to saline, as wasmeasured ex vivo by cholera toxin B binding (FIG. 13A). AIBP-imposedlipid raft reductions in spinal microglia in LPS-treated animalsaveraged 14%. We hypothesize that this moderate change in membranemicrodomain organization is sufficient to physiologically inhibitTLR4-mediated neuroinflammation and neuropathic pain, but would notinterfere with normal neuronal function.

To test this hypothesis, mice were injected with i.t. saline orrecombinant AIBP, followed by the i.t. injection of LPS, and tested forallodynia. We have earlier demonstrated that allodynia response to i.t.LPS in female mice is less robust than in males (22). Indeed, i.t. LPSinduced only partial reduction in the tactile threshold level in femalemice, and AIBP was ineffective in the early stages of allodynia,although the recovery phase trended faster in i.t. AIBP animals comparedto i.t. saline (FIG. 14).

In contrast to females, LPS induced severe tactile allodynia in malemice, which was expected from our previous experiments (19, 22).Remarkably, the initial i.t. injection of AIBP significantly preventedLPS-induced allodynia, whereas injections of denatured, heat-inactivatedAIBP had no antagonistic effect (FIGS. 13A, 13B).

Next, we compared the therapeutic effect of AIBP with that of abeta-cyclodextrin (βCD). βCDs are heptamer cyclic oligosaccharides,commonly used to solubilize hydrophobic molecules in pharmaceuticformulations as well as to remove membrane cholesterol in cell cultureexperiments and thus serving as a method to disrupt lipid rafts. βCDsinhibit TLR4-mediated inflammatory responses in vitro (23, 24). I.t. BCDprevented LPS-induced allodynia for up to 4 hours, but unlike AIBP, BCDwas not effective at 24 and 48 hour time points (FIG. 13B). Because ofthe differences in the response to i.t. AIBP between male and femalemice, we focused in subsequent studies on male mice.

We observed no motor deficits in animals injected with i.t. AIBP.Animals were systematically characterized for symmetrical gait, hindlimb weight bearing, pinnae and blink reflexes and hind paw placing andstepping (P/S). P/S assesses the integrity of light touch (Aβ afferentsfrom dorsum of paw) and the intact spinally mediated reflex involvingplantar placement and spreading of the digits, reflecting fine motorcontrol of ankle and paw. All of these measures (except pinnae andblink) are depressed or lost in a dose dependent fashion after i.t.local anesthetics and botulinum toxin (25, 26), but were unaffected ini.t. AIBP injected animals, attesting to intact motor function.

Our studies demonstrated a novel mechanism and role for AIBP inpreventing neuropathic pain. However, from the clinical perspective,reversal of chronic pain would have much greater health benefit foraffected populations. To test the therapeutic potential of AIBP inreversing neuropathic pain, we first i.t. injected male mice with LPS,followed after 24 hours by i.t. saline or AIBP. After i.t. LPS, micedeveloped severe allodynia. However, the allodynia was markedlyattenuated by a single injection of AIBP, but not saline (FIG. 13C).

In a different model of chronic pain, intraplantar injection of formalinyields acute flinching, a coordinated, high frequency extension andflexion of the injected hind paw, and after a 7 day delay, a persistenttactile allodynia progressively develops along with an associatedactivation of spinal microglial (27). In this model, we subjected malemice to i.t. injections of saline or AIBP, followed by intraplantarinjection of formalin. AIBP had no effect upon phase 1 or 2formalin-evoked hind paw flinching (FIG. 15). An unaffected acuteflinching behavior after i.t. AIBP again attests to normal motorfunction. However, AIBP reduced the allodynia otherwise observed on the7th day after administration of formalin (FIG. 13D). In a differentexperiment, mice first received intraplantar formalin and 7 days lateri.t. AIBP or saline. AIBP but not saline significantly reversedallodynia (FIG. 13E). These results demonstrate that AIBP controls thedevelopment of chronic pain following acute injury.

In rodents, as in people, the chemotherapeutic cisplatin induceslong-lasting tactile allodynia. A single i.t. AIBP injectionsignificantly reversed established cisplatin-induced allodynia, and asecond injection produced longer efficacy (FIG. 13F).

To the best of our knowledge, there is no current therapy that reversesestablished chemotherapy-induced peripheral neuropathy (CIPN) (28-30).The therapeutic effect and the absence of side effects observed in AIBPtreated mice validates and demonstrates the novel approach to treatmentof CIPN in patients as provided herein.

The need for neuraxial delivery in humans does not constitute a barrierfor developing AIBP therapy. Intrathecal therapy has significantprecedent, particularly for specific groups of patients including thosewith CIPN (31). The case has been made that the intrathecal route is auseful and efficacious route of administration for certain therapeuticproteins, peptides and oligonucleotides as opposed to small molecules(32-34). Additionally, in alternative embodiments, for AIBP therapy asprovided herein, AIPB is delivered to the CNS using moieties that targetand/or allow entry into the CNS, e.g., allow therapeutic amounts of AIBPto pass through the BBB and enter the CNS.

Noting that this invention is not limited by any particular mechanism ofaction, this work focused on AIBP-mediated inhibition of TLR4localization to lipid rafts, TLR4 dimerization, signaling and expressionof inflammatory cytokines. Similar to i.t. administration of βCD, achemical disrupting lipid rafts, i.t. AIBP prevented TLR4-mediatedallodynia in mice (FIG. 13B). However, unlike βCD, AIBP inhibition ofallodynia was sustained, which might be because in contrast toindiscriminate disruption of lipid rafts by βCD, raising spinal AIBPlevels amplifies a natural mechanism controlling TLR4 occupancy in lipidrafts. AIBP is present in the cerebrospinal fluid (16).

FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F illustratedata showing that intrathecal AIBP prevents and reverses allodynia. A,Male mice were given an i.t. injection of AIBP (0.5 μg/5 μl) or saline(5 μl); two hours later, all mice were given an i.t. injection of LPS(0.1 μg/5 μl) and were terminated 15 min later. Spinal cords wereisolated, demyelinated, stained for CD11b and cholera toxin B (CTB) andsubjected to a flow cytometry analysis. Mean±SEM; n=11; *, p<0.05(Student's t-test). FIG. 13A-13B, following baseline von Frey thresholdtesting, male mice were given an i.t. injection of AIBP (0.5 μg/5 μl),heat-inactivated hi-AIBP (0.5 μg/5 μl), beta-cyclodextrin (5 μl of 10%solution in saline), or saline (5 μl). Two hours later, all mice weregiven an i.t. injection of LPS (0.1 μg/5 μl) and tested over time fortactile allodynia; Mean±SEM; n=4-6; *, p<0.01; ***, p<0.0001 (two-wayANOVA with Bonferroni post-test). FIG. 13C, Following baseline von Freythreshold testing, male mice were given i.t. LPS (0.1 μg/5 μl).Twenty-four hours later, mice received i.t. AIBP (0.5 μg/5 μl) or saline(5 μl). Mean±SEM; n=4 per group; *, p<0.05 (Student's t-test for the 48hour time point only). FIG. 13D, Following baseline von Frey thresholdtesting, male mice were given an intraplantar injection of formalin inone hind paw. The graph shows $baseline-normalized changes in thewithdrawal threshold in the ipsilateral paw. Mean±SEM; n=4-12 per group;*, p<0.05 (Student's t-test for the 7 day time point only). FIG. 13E, Ina different group of male mice, von Frey readings were #normalized atthe 7th day post-formalin and the mice received i.t. AIBP (0.5 μg/5 μl)or saline (5 μl). Mean±SEM; n=4 per group; *, p<0.05 between 0 and 24hours (repeated measures one-way ANOVA with Bonferroni post-test). FIG.13F, Male mice (n=18) received 6 i.p. injections of cisplatin (2.3mg/kg) over a period of 11 days (gray shaded box) to establishallodynia. On day 25, twelve mice were treated with 1st i.t. AIBP (0.5μg/5 μl; red circles) and six mice with i.t. saline (white squares). Onday 30, mice received 2nd i.t. injection: 1) six mice that received AIBPwere injected with AIBP (red circles); 2) six mice that received AIBPwere injected with saline (dark grey squares); and 3) mice receivingsaline were injected with saline (white squares). Mean±SEM. *, p<0.05;***, p<0.001; ****, p<0.0001 (two-way ANOVA with Bonferroni post-test).

FIG. 14 graphically illustrates data from studies of Intrathecalinjections of AIBP and LPS in female mice. Following baseline von Freythreshold testing, female mice were given an i.t. injection of AIBP (0.5μg/5 μl) or saline (5 μl). Two hours later, all mice were given an i.t.injection of LPS (0.1 μg/5 μl) and tested over time for tactileallodynia. Mean±SEM; n=4; no statistically significant differences(two-way ANOVA with Bonferroni post-test).

FIG. 15 graphically illustrates data from studies showing thatIntrathecal AIBP does not affect acute post-formalin paw flinching.Animals were given an intraplantar injection of formalin in one hindpaw. The graph shows total numbers of hind paw flinches in phase I (1-9min) and phase II (10-50 min). Mean±SD; n=4-12 per group; #,non-significant, p>0.05 (Student's t-test).

Example 3: Efficacy Demonstrated in Exemplary Methods—AIBP Treats andPrevents Neurodegeneration and Neurodegenerative Diseases or Conditions

This example describes and demonstrates exemplary embodiments, and theefficacy of methods as provided herein to e.g., to treat, prevent,decrease the severity of or ameliorate neurodegeneration and/orneuroinflammation, including e.g., a neurodegenerative disease such asAlzheimer's disease, chronic traumatic encephalopathy (CTE) or CNS ornerve damage due to trauma, such as traumatic brain injury (TBI).

Our studies, using both in vitro systems and animal models, havedemonstrated that AIBP reduces pathologic lipid rafts and the occupancyof inflammatory receptors (TLR4) in lipid rafts; see e.g., ref [1] andFIGS. 1 and 11 to 14, and discussions, above. See also FIG. 16, whichschematically and graphically illustrates that AIBP reduces TLR4occupancy in microglia lipid rafts. BV-2 microglia cells were incubatedfor 2 hours with vehicle (0.1% BSA) or 0.2 μg/ml AIBP (in 0.1% BSA) inserum-containing medium and stimulated with 10 ng/ml LPS. TLR4 occupancyin lipid rafts was tested 15 min after adding LPS. Mean±SEM; n=6; *,p<0.05; **, p<0.01 (Student's t-test).

FIG. 17 graphically illustrates data showing that AIBP reduces lipidrafts in vivo in spinal cord microglia. Male mice were given an i.t.injection of AIBP (0.5 μg/5 μl) or saline (5 up; two hours later, allmice were given an i.t. injection of LPS (0.1 μg/5 μl) and wereterminated 15 min later. Spinal cords were isolated, demyelinated,stained for CD11b and cholera toxin B (CTB) and subjected to a flowcytometry analysis, FIG. 17A, results graphically displayed in FIG. 17B.Mean±SEM; n=11; *, p<0.05 (Student's t-test).

FIG. 18 schematically and graphically illustrates that AIBP reducesinflammatory responses in microglia. FIG. 18B-C graphically illustratedata where BV-2 microglia cells were incubated for 2 hours with vehicle(0.1% BSA) or 0.2 μg/ml AIBP (in 0.1% BSA) in serum-containing mediumand stimulated with 10 ng/ml LPS. p65 and ERK1/2 phosphorylation weretested after 30 min (immunoblot illustrated in FIG. 18A, and resultsgraphically illustrated in FIG. 18B), and cytokine mRNA expression after2 h of incubation with LPS (FIG. 18C). FIG. 18D graphically illustratesdata where primary mouse microglia (pooled from 5-6 mice per sample)were incubated for 2 hours with vehicle (0.1% BSA) or 0.2 μg/ml AIBP (in0.1% BSA) in serum-containing medium and stimulated with 10 ng/ml LPSfor 1 hour. Mean±SEM; n=4-6 for BV-2; n=3 for primary microgliaexperiments; *, p<0.05; **, p<0.01; ***, p<0.005 (Student's t-test).

FIG. 19 graphically illustrates data showing that AIBP reducesneuroinflammation in the CNS. Male mice were given an i.t. injection ofAIBP (0.5 μg/5 μl) or saline (5 up; and two hours later, mice were givenan i.t. injection of LPS (0.1 μg/5 μl). Four hours later, mice weresacrificed, cerebrospinal fluid (CSF) was collected and analyzed inELISA to determine levels of inflammatory cytokines IL-1 (FIG. 19A),TNF-alpha (TNF-α) (FIG. 19B) and IL-1beta (IL-1β) (FIG. 19C). Pooled CSFsamples from 4 mice in each group.

Thus, these data demonstrate that compositions and methods providedherein can be used to treat, prevent, decrease the severity of orameliorate numerous pathological processes associated with pathologiclipid rafts and the occupancy of inflammatory receptors (TLR4) in lipidrafts, including neuroinflammation, Alzheimer's diseases (AD) and otherneurodegenerative diseases or conditions. These include but not limitedto γ-secretase cleavage of APP, apoE lipidation, and neuroinflammation[2-4]. Our studies of neuropathic pain have demonstrated that AIBPeffectively reduces inflammatory responses in microglia (see e.g., FIG.12 and discussion above) and that spinal delivery of AIBP reducesneuroinflammation in the CNS in the mouse model (FIG. 19).

Thus, we propose that raising AIBP levels in the CNS will prevent, delayand/or reverse the development of neurodegenerative diseases orconditions. AIBP can be delivered to the CNS via adeno-associated virus(AAV; see the accompanying document) or other CNS delivery methods,e.g., a CNS targeted vehicle capable of penetrating the blood brainbarrier (BBB) as discussed above, such as e.g., with Clostridialneurotoxins-derived carriers for retroaxonal delivery of cargo proteinsand genes to the brain [5].

Example 4: Efficacy Demonstrated in Exemplary Methods—AIBP Delivery tothe CNS with AAV to Treat Neurodegeneration and NeurodegenerativeDiseases

This example describes and demonstrates the exemplary embodiment ofdelivery of AIBP to the CNS to e.g., treat, prevent, decrease theseverity of or ameliorate neurodegeneration and/or neuroinflammation,including e.g., a neurodegenerative disease or condition such asAlzheimer's disease, chronic traumatic encephalopathy (CTE) or CNS ornerve damage due to trauma, such as traumatic brain injury (TBI).

The method of AIBP delivery used in preclinical (mouse) studies was:intrathecal injections of recombinant AIBP protein (29 kDa). Inalternative embodiment, this AIBP is modified, e.g., by introducingmimetic peptides or other derivatives.

In alternative embodiments, AIPB is delivered to the CNS by intravenous(IV) injections of a large dose of AIBP or its derivatives designed tocross the blood-brain barrier. For example, in alternative embodiments,AIBP polypeptide, or AIBP encoding nucleic acid, e.g., nucleic acidcontained in a vector, is carried in a nanoparticle, a particle, amicelle or a liposome or lipoplex, a polymersome, a polyplex or adendrimer, which optionally can further comprise or express a cellpenetrating moiety or peptide, and/or a CNS targeting moiety or peptide.

In one alternative embodiment, AIBP is delivered to the CNS using anadeno-associated virus (AAV); optionally, as a single dose (optionally asingle dose of about 10¹⁰-10¹⁴ gc/kg) of AAV, or multiple dosages ofAAV, as needed, which will ensure sustained secretion of AIBP in thebrain. In alternative embodiments, sufficient levels of individualdosages, or multiple administrations, are given to a patient in needthereof to achieve sustained levels of AIBP in the CNS, which can beadvantageous in the treatment of a neurodegenerative disease or otherchronic CNS condition.

For preclinical studies, we first made mouse AIBP-AAV. Specifically, weused commercially available AAV-DJ/8, which has been reported topreferentially infect CNS cells [1-3]. Similar strategy can be used toproduce human AIBP-AAV for clinical applications, using other availableor proprietary AAV constructs.

Virus Production and Testing

Mouse AIBP (24-863 aa) was fused with fibronectin secretion sequence(FIB) at the N-terminus and 6×-His at the C-terminus (FIB-mAIBP-His).FIB-mAIBP-His or FIB-GFP-His was cloned into the pAAV-MCS vector, asillustrated in FIG. 20. All clones were sequenced to confirm thepresence of the insert.

AAV-293™ cells (Agilent Technologies) were transfected with 20 μg ofeach pAAV-FIB-mAIBP-His, pAAV-DJ/8 (Cell Biolabs), and pHelper™ DNA(Cell Biolabs) following the routine calcium phosphate-based protocol(Agilent Technologies). Subsequent steps of virus harvest, purificationand storage were according to the manufacturer's protocol (AgilentTechnologies).

Viral DNA was extracted from purified virus and the number of genecopies (gc) was determined using qPCR with primers for the invertedterminal repeats (ITRs).

To test FIB-mAIBP-His-AAV-DJ/8, we infected HEK 293 cells. Three dayslater cells were harvested and mAIBP-His expression was confirmed inwestern blot with an anti-His antibody, as illustrated in FIG. 21: mAIBPexpression in HEK293 cells infected with FIB-mAIBP-His-AAV-DJ/8. Theblot was probed with an anti-His antibody. The two bands represent mAIBPwith the fibronectin secretion sequence still attached (upper band) andmAIBP processed for secretion from which the fibronectin secretionsignal was cleaved off (lower band). The cells were infected with3.3×10″ gc (sample 1), 6.6×10¹¹ gc (sample 1), 9.9×10¹¹ gc (sample 1),2.2×10¹¹ gc (sample 1), 4.4×10¹¹ gc (sample 1), and 6.6×10¹¹ gc (sample1), and analyzed 3 days later. Non-infected cells were used as anegative control.

SEQ ID NO:2 and SEQ ID NO:3 are the nucleotide and protein sequences,respectively, of murine AIBP supplemented with the fibronectin secretionsignal (italic) at the N-terminus, and with the His tag (underlined) atthe C-terminus; the product is abbreviated as FIB-mAIBP-His:

SEQ ID NO: 2: ATG CTC AGG GGT CCG GGA CCC GGG CGG CTG CTG CTGCTA GCA GTC CTG TGC CTG GGG ACA TCG GTG CGC TGCACC GAA ACC GGG AAG AGC AAG AGGCAGCAGAGTGTGTGTCGTGCAAGGCCCATCTGGTGGGGAACACAGCGCCGGGGCTCGGAGACCATGGCGGGCGCTGCGGTGAAGTACTTAAGTCAGGAGGAGGCTCAGGCCGTGGACCAAGAGCTTTTTAACGAGTATCAGTTCAGCGTGGATCAACTCATGGAGCTGGCCGGGTTGAGCTGTGCCACGGCTATTGCCAAGGCTTATCCCCCCACGTCTATGTCCAAGAGTCCCCCGACTGTCTTGGTCATCTGTGGCCCCGGAAATAACGGAGGGGATGGGCTGGTCTGTGCGCGACACCTCAAACTTTTTGGTTACCAGCCAACTATCTATTACCCCAAAAGACCTAACAAGCCCCTCTTCACTGGGCTAGTGACTCAGTGTCAGAAAATGGACATTCCTTTCCTTGGTGAAATGCCCCCAGAGCCCATGATGGTGGACGAGCTGTATGAGCTGGTGGTGGACGCCATCTTCGGCTTCAGTTTCAAGGGTGACGTTCGGGAGCCATTCCACAGCATCCTGAGTGTCTTGAGTGGACTCACTGTGCCCATTGCTAGCATCGACATTCCCTCAGGATGGGATGTAGAGAAGGGAAACCCTAGCGGAATCCAACCAGACTTACTCATCTCACTGACGGCACCCAAGAAGTCTGCAACTCACTTTACTGGCCGATATCATTACCTTGGGGGTCGCTTTGTACCACCTGCTCTAGAGAAGAAGTACCAGCTGAACCTGCCATCTTACCCTGACACAGAGTGTGTCTACCGTCTACAGCATCATCATCATCATCATTAA SEQ ID NO: 3:MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKRQQSVCRARPIWWGTQRRGSETMAGAAVKYLSQEEAQAVDQELFNEYQFSVDQLMELAGLSCATAIAKAYPPTSMSKSPPTVLVICGPGNNGGDGLVCARHLKLFGYQPTIYYPKRPNKPLFTGLVTQCQKMDIPFLGEMPPEPMMVDELYELVVDAIFGFSFKGDVREPFHSILSVLSGLTVPIASIDIPSGWDVEKGNPSGIQPDLLISLTAPKKSATHFTGRYHYLGGRFVPPALEKKYQLNLPSYPDTECVYRLQHHHHHH

In alternative embodiment, human AIBP polypeptide, or a nucleic acidencoding an AIBP is administered to an individual in need thereof, or isused to manufacture a formulation or pharmaceutical, or is used to makea vector or expression vehicle for administration:

Human AIBP-encoding nucleic acid (cDNA) (SEQ ID NO: 4)GGGCCGGGCCGGGCCGGGGGCGCGCGCTCTGCGAGCTGGATGTCCAGGCTGCGGGCGCTGCTGGGCCTCGGGCTGCTGGTTGCGGGCTCGCGCGTGCCGCGGATCAAAAGCCAGACCATCGCCTGTCGCTCGGGACCCACCTGGTGGGGACCGCAGCGGCTGAACTCGGGTGGCCGCTGGGACTCAGAGGTCATGGCGAGCACGGTGGTGAAGTACCTGAGCCAGGAGGAGGCCCAGGCCGTGGACCAGGAGCTATTTAACGAATACCAGTTCAGCGTGGACCAACTTATGGAACTGGCCGGGCTGAGCTGTGCTACAGCCATCGCCAAGGCATATCCCCCCACGTCCATGTCCAGGAGCCCCCCTACTGTCCTGGTCATCTGTGGCCCGGGGAATAATGGAGGAGATGGTCTGGTCTGTGCTCGACACCTCAAACTCTTTGGCTACGAGCCAACCATCTATTACCCCAAAAGGCCTAACAAGCCCCTCTTCACTGCATTGGTGACCCAGTGTCAGAAAATGGACATCCCTTTCCTTGGGGAAATGCCCGCAGAGCCCATGACGATTGATGAACTGTATGAGCTGGTGGTGGATGCCATCTTTGGCTTCAGCTTCAAGGGCGATGTTCGGGAACCGTTCCACAGCATCCTGAGTGTCCTGAAGGGACTCACTGTGCCCATTGCCAGCATCGACATTCCCTCAGGATGGGACGTGGAGAAGGGAAATGCTGGAGGGATCCAGCCAGACTTGCTCATATCCCTCACAGCCCCCAAAAAATCTGCAACCCAGTTTACCGGTCGCTACCATTACCTGGGGGGTCGTTTTGTGCCACCTGCTCTGGAGAAGAAGTACCAGCTGAACCTGCCACCCTACCCTGACACCGAGTGTGTCTATCGTCTGCAGTGAGGGAAGGTGGGTGGGTATTCTTCCCAATAAAGACTTAGAGCCCCTCTCTTCCAGAACTGTGGATTCCTGGGAGCTCCTCTGGCAATAAAAGTCAGTGAATGGTGGAAGTCAGAGACCAACCCTGGGGATTGGGTGCCATCTCTCTAGGGGTAACACAAAGGGCAAGAGGTTGCTATGGTATTTGGAAACAATGA AAATGGACTGTTAGATGCCAAHuman AIBP polypeptide (SEQ ID NO: 5)MSRLRALLGLGLLVAGSRVPRIKSQTIACRSGPTWWGPQRLNSGGRWDSEVMASTVVKYLSQEEAQAVDQELFNEYQFSVDQLMELAGLSCATAIAKAYPPTSMSRSPPTVLVICGPGNNGGDGLVCARHLKLFGYEPTIYYPKRPNKPLFTALVTQCQKMDIPFLGEMPAEPMTIDELYELVVDAIFGFSFKGDVREPFHSILSVLKGLTVPIASIDIPSGWDVEKGNAGGIQPDLLISLTAPKKSATQFTGRYHYLGGRFVPPALEKKYQLNLPPYPDTECVYRLQ

In one embodiment, a secretion signal is added to ensure robustsecretion of AIBP, for example, a fibronectin secretion signal is addedto N terminus of AIBP (see italicized sequences in SEQ ID NO:2 and SEQID NO:3); or a nucleic acid encoding a secretion signal is added to theAIBP coding sequence. In alternative embodiments, a secretion signal isa fibronectin secretion signal, an immunoglobulin heavy chain secretionsignal or an immunoglobulin kappa light chain secretory peptide (see,e.g., PLoS One. 2015; 10(2): e0116878), or an interleukin-2 signalpeptide (see, e.g., J. Gene Med. 2005 March; 7(3):354-65).

In alternative embodiments, the polypeptide coding sequences areoperatively linked to a promoter, e.g., a constitutive, inducible,tissue specific (e.g., nerve or brain tissue specific) or ubiquitouspromoter or other transcriptional activating agent.

Example 5: Efficacy Demonstrated in Exemplary Methods—Intravenous (IV)Administration of AIBP Ameliorate Migraines

This example describes and demonstrates, using an art-accepted animal(murine) model for migraines, the exemplary embodiment of delivery ofAIBP for the treatment or amelioration of migraines.

Compound 48/80, a condensation product ofN-methyl-p-methoxyphenethylamine with formaldehyde, promotes mast cellsdegranulation, release of histamine, and induces migraine-like lightaversion in mice (Pain. 2007 July; 130(1-2):166-76). This is tested in ahousing unit with connected light and dark chambers, and time spent ineach chamber is recorded. Unchallenged mice spend approximately 50% timein each chamber, but mice treated with compound 48/80 prefer the darkchamber for at least 2 hours, and then slowly recover.

Male and female mice were injected i.v. with AIBP (0.5 μg/100 μl) orsaline (100 μl) 2 hours prior to i.p. injection of compound 48/80 (2mg/kg). AIBP-injected mice did not develop light aversion (FIGS. 22 and23), thus demonstrating the efficacy administration of AIBP for thetreatment or amelioration of migraines.

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Free Cholesterol    Accumulation in Macrophage Membranes Activates Toll-Like Receptors    and p38 Mitogen-Activated Protein Kinase and Induces Cathepsin K.    Circ Res. 2009; 104:455-465-   37. Mineo C, Shaul P W. Regulation of signal transduction by HDL. J    Lipid Res. 2013; 54:2315-2324-   38. Terasaka N, Yu S, Yvan-Charvet L, Wang N, Mzhavia N, Langlois R,    Pagler T, Li R, Welch C L, Goldberg I J, Tall A R. ABCG1 and HDL    protect against endothelial dysfunction in mice fed a    high-cholesterol diet. The Journal of Clinical Investigation. 2008;    118:3701-3713-   39. van der Westhuyzen D R, de Beer F C, Webb N R. HDL cholesterol    transport during inflammation. Curr. Opin. Lipidol. 2007; 18:147-151-   40. Murphy A J, Woollard K J, Hoang A, Mukhamedova N, Stirzaker R A,    McCormick S P A, Remaley A T, Sviridov D, Chin-Dusting J.    High-Density Lipoprotein Reduces the Human Monocyte Inflammatory    Response. Arterioscler Thromb Vasc Biol. 2008; 28:2071-2077-   41. 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A dural lymphatic vascular system that drains    brain interstitial fluid and macromolecules. The Journal of    Experimental Medicine. 2015-   47. Martel C, Li W, Fulp B, Platt A M, Gautier E L, Westerterp M,    Bittman R, Tall A R, Chen S H, Thomas M J, Kreisel D, Swartz M A,    Sorci-Thomas M G, Randolph G J. Lymphatic vasculature mediates    macrophage reverse cholesterol transport in mice. The Journal of    Clinical Investigation. 2013; 123:1571-1579-   48. Hobbs H H, Rader D J. ABC1: connecting yellow tonsils,    neuropathy, and very low HDL. J Clin Invest. 1999; 104:1015-1017-   49. van Exel E, de Craen A J, Gussekloo J, Houx P, Bootsma-van der    Wiel A, Macfarlane P W, Blauw G J, Westendorp R G. Association    between high-density lipoprotein and cognitive impairment in the    oldest old. Ann Neurol. 2002; 51:716-721-   50. Merched A, Xia Y, Visvikis S, Serot J M, Siest G. 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Genomics. 2002; 79:693-702-   54. Jha K N, Shumilin I A, Digilio L C, Chertihin O, Zheng H,    Schmitz G, Visconti P E, Flickinger C J, Minor W, Herr J C.    Biochemical and structural characterization of apolipoprotein A-I    binding protein, a novel phosphoprotein with a potential role in    sperm capacitation. Endocrinology. 2008; 149:2108-2120-   55. Rudolph C, Sigruener A, Hartmann A, Orso E, Bals-Pratsch M,    Gronwald W, Seifert B, Kalbitzer H R, Verdorfer I, Luetjens C M,    Ortmann O, Bornstein S R, Schmitz G. ApoA-I-binding protein (AI-BP)    and its homologues hYjeF_N2 and hYjeF_N3 comprise the YjeF_N domain    protein family in humans with a role in spermiogenesis and    oogenesis. Horm. Metab Res. 2007; 39:322-335-   56. Marbaix A Y, Noel G, Detroux A M, Vertommen D, Van Schaftingen    E, Linster C L. Extremely Conserved ATP- or ADP-dependent Enzymatic    System for Nicotinamide Nucleotide Repair. J Biol Chem. 2011;    286:41246-41252-   57. 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Systemic TAK-242 prevents intrathecal LPS    evoked hyperalgesia in male, but not female mice and prevents    delayed allodynia following intraplantar formalin in both male and    female mice: The role of TLR4 in the evolution of a persistent pain    state. Brain Behav Immun. 2016; 56:271-80.-   23. Meng G, et al. Emodin suppresses lipopolysaccharide-induced    pro-inflammatory responses and NF-kappaB activation by disrupting    lipid rafts in CD14-negative endothelial cells. Br J Pharmacol.    2010; 161(7):1628-44.-   24. Shridas P, et al. Group X secretory phospholipase A2 enhances    TLR4 signaling in macrophages. J Immunol. 2011; 187(1):482-9.-   25. Penning J P, and Yaksh T L. Interaction of intrathecal morphine    with bupivacaine and lidocaine in the rat. Anesthesiology. 1992;    77(6):1186-2000.-   26. Huang P P, et al. Spinal botulinum neurotoxin B: effects on    afferent transmitter release and nociceptive processing. PLoS One.    2011; 6(4):e19126.-   27. Wu Y, et al. 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REFERENCES—EXAMPLE 3

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REFERENCES—EXAMPLE 4

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A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for treating, ameliorating, reversing ordecreasing the severity or duration of a primary headache, wherein themethod comprises: (a) providing or having provided a formulation or apharmaceutical composition comprising: an ApoA-I Binding Protein(APOA1BP) polypeptide compound or composition; and (b) administering orhaving administered the formulation or the pharmaceutical composition of(a) to a subject in need thereof, thereby treating, ameliorating,reversing or decreasing the severity or duration of the primaryheadache, or (b) administering the formulation or the pharmaceuticalcomposition comprising: an ApoA-I Binding Protein (APOA1BP) polypeptidecompound or composition to a subject in need thereof, thereby treating,ameliorating, reversing or decreasing the severity or duration of theprimary headache.
 2. The method of claim 1, wherein the primary headacheis a migraine.
 3. The method of claim 1, wherein the primary headache isa cluster headache.
 4. The method of claim 1, wherein the ApoA-I BindingProtein (APOA1BP) polypeptide or protein is a mammalian APOA1BPpolypeptide or peptide.
 5. The method of claim 4, wherein the ApoA-IBinding Protein (APOA1BP) polypeptide or protein is a human APOA1BPpolypeptide or peptide.
 6. The method of claim 1, wherein the subject isa human.
 7. The method of claim 1, wherein the subject is an animal. 8.The method of claim 1, wherein the APOA1BP polypeptide or peptide is arecombinant APOA1BP polypeptide or peptide having an APOA1BP activity.9. The method of claim 1, wherein the APOA1BP polypeptide or peptide isa synthetic APOA1BP polypeptide or peptide.
 10. The method of claim 1,wherein the APOA1BP polypeptide or peptide is a APOA1BPactivity-stimulating compound or composition.
 11. The method of claim 1,wherein the formulation or pharmaceutical composition is formulated foradministration in vivo; or is formulated for enteral or parenteraladministration.
 12. The method of claim 1, wherein the formulation orpharmaceutical composition is formulated for administration by in vivointrathecal injection.
 13. The method of claim 1, wherein theformulation or pharmaceutical composition is formulated for oraladministration.
 14. The method of claim 1, wherein the formulation orpharmaceutical composition is formulated for intravenous (IV)administration.
 15. The method of claim 1, wherein the APOA1BPpolypeptide or peptide or the formulation or pharmaceutical composition,is formulated in or with a nanoparticle, a particle, a micelle or aliposome or lipoplex, a polymersome, a polyplex or a dendrimer.
 16. Themethod of claim 1, wherein the APOA1BP polypeptide or peptide or theformulation or pharmaceutical composition, is formulated in or as ananoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab,a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, alozenge, an aqueous or a sterile or an injectable solution, or animplant.
 17. The method of claim 16, wherein the implant is anintrathecal implant.
 18. The method of claim 16, wherein thenanoparticle, particle, micelle or liposome or lipoplex, polymersome,polyplex or dendrimer further comprise or express a cell or CNSpenetrating moiety or peptide or a CNS targeting moiety or peptide.